In this study, tandem MS analysis of the GC-A receptor, expressed in the human embryonic kidney cell line HEK293, revealed unambiguously that the intracellular domain of the receptor is phosphorylated at multiple residues: Ser487, Ser497, Thr500, Ser502, Ser506, Ser510 and Thr513. MS quantification based on multiple reaction monitoring demonstrated that ANP-provoked desensitization was accompanied by a complex pattern of receptor phosphorylation and dephosphorylation. The population of completely phosphorylated GC-A was diminished. However, intriguingly, the phosphorylation of GC-A at Ser487 was selectively enhanced after exposure to ANP. The functional relevance of this observation was analysed by site-directed mutagenesis. The substitution of Ser487 by glutamate (which mimics phosphorylation) blunted the activation of the GC-A receptor by ANP, but prevented further desensitization. Our data corroborate previous studies suggesting that the responsiveness of GC-A to ANP is regulated by phosphorylation. However, in addition to the dephosphorylation of the previously postulated sites , homologous desensitization seems to involve the phosphorylation of GC-A at Ser487, a newly identified site of phosphorylation. The identification and further characterization of the specific mechanisms involved in the downregulation of GC-A responsiveness to ANP may have important pathophysiological implications.Atrial natriuretic peptide (ANP), via its guanylyl cyclase A (GC-A) receptor and intracellular guanosine 3′,5′-cyclic monophosphate production, is critically involved in the regulation of blood pressure. In patients with chronic heart failure, the plasma levels of ANP are increased, but the cardiovascular actions are severely blunted, indicating a receptor or postreceptor defect. Studies on metabolically labelled GC-A-overexpressing cells have indicated that GC-A is extensively phosphorylated, and that ANP-induced homologous desensitization of GC-A correlates with receptor dephosphorylation, a mechanism which might contribute to a loss of function Structured digital abstractMINT-7713870, MINT-7713887: PMCA (uniprotkb:P20020) and GC-A (uniprotkb:P18910) colocalize (MI:0403) by fluorescence microscopy (MI:0416)• ANP and BNP gene loci with circulating concentrations of ANP/BNP and blood pressure , and the lysates were pelleted by centrifugation . The protein concentration was determined by bicinchoninic acid protein assay. All guanylyl cyclase activity assays were carried out at 37 °C in 50 mm Hepes buffer, pH 7.4, containing 50 mm NaCl, 5% glycerol, 0.05% BSA, 1 mm 3-isobutyl-1-methylxanthine, 2 mm GTP, 30 mm creatine phosphate and 1.5 units·mL−1 (0.3 units per assay) creatine phosphokinase [2+ GTP and ATP , 0.1–100 nm ANP, ATP and Mg2+ GTP (stimulated activity) or Triton X-100 and Mn2+ GTP (detergent-stimulated activity) for 10 min [Rat FLAG-tagged GC-A in pCMV5 vector served as template for the site-directed substitution of the novel phosphorylation site Ser487 with glutamate. The oligonuclotide primers and details of PCRs are depicted in hokinase ,17,21,22r 10 min ,17,21,22r 10 min . Triton r 10 min ,17,21,22t-test (P < 0.05). Data are given as the mean ± SE.Statistical comparisons were performed using Student’s


Arabidopsis thaliana, and test crosstalk between CPK3 and the major salt-stress activated MAPKs MPK4 and MPK6 in the salt stress response. CPK3 kinase activity was induced by salt and other stresses after transient overexpression in Arabidopsis protoplasts, but endogenous CPK3 appeared to be constitutively active in roots and leaves in a strictly Ca2+-dependent manner. cpk3 mutants show a salt-sensitive phenotype comparable with mutants in MAPK pathways. In contrast to animal cells, where crosstalk between Ca2+ and MAPK signalling is well established, CPK3 seems to act independently of those pathways. Salt-induced transcriptional induction of known salt stress-regulated and MAPK-dependent marker genes was not altered, whereas post-translational protein phosphorylation patterns from roots of wild type and cpk3 plants revealed clear differences. A significant portion of CPK3 was found to be associated with the plasma membrane and the vacuole, both depending on its N-terminal myristoylation. An initial proteomic study led to the identification of 28 potential CPK3 targets, predominantly membrane-associated proteins.Plants use different signalling pathways to respond to external stimuli. Intracellular signalling via calcium-dependent protein kinases (CDPKs) or mitogen-activated protein kinases (MAPKs) present two major pathways that are widely used to react to a changing environment. Both CDPK and MAPK pathways are known to be involved in the signalling of abiotic and biotic stresses in animal, yeast and plant cells. Here, we show the essential function of the CDPK CPK3 (At4g23650) for salt stress acclimation in  In order to test whether CPK3 is transcriptionally regulated in response to salt stress, we performed semi-quantitative RT-PCR using CPK3-specific primers and actin  epitope-tagged CPK3 transiently in Arabidopsis protoplasts, and measured its kinase activity towards histone III-S as the substrate in immunocomplex kinase assays 15 min after the application of different stimuli. CPK3 kinase activity was found to be induced by almost all treatments performed, but clearly the strongest activation was detected after salt-stress treatment . To veriin roots , leaves in roots  in plantCPK3 were obtained from the Salk collection   (cpk3 mutants and overexpressor lines with the corresponding wild type on agar plates containing 150 mm NaCl (m NaCl a clear difference appeared. Germination of both cpk3 mutant lines (cpk3-2 and cpk3-1) was severely impaired compared with the wild type, whereas the germination rate of the CPK3 overexpressing line (cpk3-3) was clearly increased. Moreover, the increase in the germination rate on salt corresponded with the level of CPK3 expression, as revealed by the analysis of different 35S::CPK3-YFP lines  . These l mm NaCl . WithoutFP lines . In the cpk3-2 knock-out line. CPK3 protein could be detected in all tissues, which corresponds to transcript levels of published microarray data , and would be consistent with older published data on CPK3 , thus aborting the N-myristoylation of CPK3. As expected, the G2A mutant was no longer membrane associated, and displayed a strong accumulation in the nucleus and the cytosolic lobes of the epidermal cells  at the vacuolar membrane , vacuolar membrane (V-ATPase), mitochondrial membranes (Porin) and the endoplasmic reticulum (ER) (Sar1)  (Sar1) . Consistcpk3 knock-out and overexpressor lines. A total of 22 target genes known to be regulated in the salt stress response, based on published microarray data (cpk3 mutant (cpk3-2), and the two CPK3 overexpressor lines CPK3-1 and CPK3-2. ERF6 and ACS6), synthesis of the compatible solutes galactinol (GolS2) and proline (P5CS), transcriptional regulation (STZ), and general stress response . It turned out that all of the selected genes were normally induced in all lines. Furthermore, the complete set of genes that we analysed in these studies included the Na+/H+ antiporters NHX1 and SOS1, the Na+-induced K+ channel KC1, several genes involved in trehalose synthesis  and proline catabolism (PDH), the ABA- and salt stress-responsive protein phosphatases AHG3 and PP2C, and the general stress-response factors ERD15 and RD29b. But also those typical salt stress marker genes were normally induced.The observed nuclear localization of CPK3 suggests a role in the transcriptional regulation of the stress response. This is known for MAPK pathways, which predominantly target transcriptional responses in animals  and planmkk2 mutants show a phenotype similar to cpk3 on salt media, and the MKK2 pathway regulates a set of 127 target genes, including typical salt stress markers  before and 30 min after the application of salt stress. All experiments were carried out three times with identical results. More than 300 protein spots could be detected reproducibly after 2D gel separation of 100 μg of total protein by Coomassie staining, after blotting on polyvinylidene difluoride (PVDF) membranes. Some differences in threonine phosphorylation were already visible between the wild type and the cpk3 mutant without stress conditions .The previous data, indicating that CPK3 activity has no influence on salt stress-dependent gene expression, gave rise to the question of how the salt-sensitive phenotype of the s tissue , we focunditions . This renditions , indicat3 mutant , and 15 3 mutant . In ordeeriments ; therefoeriments . From theriments . In orde targets . As expein planta. This apparent discrepancy might be caused simply by the strong difference in the expression level, which could affect low basal Ca2+ signals. Another explanation could be that large quantities of Ca2+ are released from the cell wall/apoplast during the preparation of the protein extracts from plant tissues, but not from protoplasts. Nevertheless, cpk3 knock-out mutants displayed a salt-sensitive phenotype, whereas CPK3 overexpression improved salt tolerance, and this effect correlated with CPK3 expression levels, as revealed by RT-PCR. Only sparse functional and in planta data on this particular CDPK have been published so far. cpk3 cpk6 double knock-out plants displayed altered responses of vacuolar potassium channels in leaf guard cells in response to ABA, but that neither a direct phosphorylation of the channel itself nor a phenotype of the knock-out plants in response to stress was exhibited. The phenotype we observed for cpk3 mutants resembled that of MAPK mutants under similar stress conditions  regulate the Ras/Raf/ERK-MAPK pathway . The early dehydration-induced gene ERD13, a glutathione-S-transferase, has been localized to the vacuole , gives the first hints about negative regulators of CPK3. The importance of negative regulation is well known from ABA signalling, and recently a pathway where three PP2C phosphatases regulate the protein kinase SnRK2.6/OST1 has been described by The identification of three PP2C-type protein phosphatases, including one with predicted N-myristoylation. We could clearly observe co-localization of CPK3-YFP with a vacuolar and a plasma membrane marker in infiltrated leaves, and a biochemical fractionation of endogenous CPK3 by two-phase partitioning confirmed its localization at the plasma membrane. Notably, this partitioning does not distinguish between remaining plasma membranes and other membranes in the lower phase, thus leaving the evidence for vacuolar localization of CPK3 based on the co-localization of the YFP fusion proteins. A similar complex localization pattern as found for CPK3 has been described for CPK32, which harbours N-myristoylation and palmitoylation sites  seeds were surface sterilized using the vapour-phase method (−1 kanamycin when required. Plants were grown under 16 h of light with 120 μmol m−2 s−1 light intensity at 25°C. Seeds were stratified by incubation in the dark at 4°C for 2 days prior to placing them in the light. Germination assays were performed on agar plates containing quarter-strength Hoagland medium supplemented with 150 mm NaCl. Germination rates (per seeds) were scored after 6 days. The hydroponic cultivation of Arabidopsis plants was performed in half-strength Hoagland medium according to the method described by e method . SterileArabidopsis protoplasts were prepared from a root suspension culture , and traCPK3 T-DNA insertion lines   or from a cDNA library  was transformed with expression constructs, cloned into the pGEX4-T1 vector . The growth of bacteria and isolation of recombinant GST fusion proteins were performed according to the method described by m reduced glutathione, 250 mm NaCl, 0.5% Triton X-100 in Tris-buffered saline (TBS) Tween, which was changed to kinase buffer  by using PD10 columns (Amersham).m Tris, pH 7.8, 75 mm NaCl, 10 mm MgCl2, 15 mm EGTA, 1 mm DTT, 1 mm NaF, 0.5 mm NaVO3, 15 mmβ-glycero-phosphate, 15 mmp-nitrophenyl-phosphate, 0.1% Tween 20, 0.5 mm phenylmethylsulfonyl fluoride, 5 μg ml−1 leupeptin, and 5 μg ml−1 aprotinin. Usually 200 mg of leaf material were ground in a 1.5-ml reaction tube in 200 μl of protein extraction buffer together with sea sand. Extracts were further clarified by centrifugation at 16 000 g for 10 min at 4°C.Proteins from all plant materials were extracted in protein extraction buffer . A 2-μg portion of plasmid template was linearized (NotI) and in vitro translated either in the presence of 10 μCi of l-[35S]methionine , or 50 μCi of -labelled myristic acid . Reaction products were separated on 10% (w/v) SDS-polyacrylamide gels, and incubated with autoradiography intensifier prior to detection on X-ray film.The coding regions of Immunocomplex kinase assays were performed according to Nicotiana tabaccum leaves were used for infiltration, as described in 2A mutant, YFP alone, TPK1 and CPK9 were cloned as KpnI–SacI fragments in the binary plant expression vector pBIN19. Proteins were transiently expressed under the control of the CaMV 35S promoter after infiltration of tobacco leaves with Agrobacteria. The pictures were taken 2 days after infiltration of developing young leaves using a Zeiss Axioplan laser scanning confocal microscope.Plasma membranes were isolated in an aqueous two-phase system according to m EDTA, 200 mm sodium acetate, pH 5.2), 130 μl phenol (pH 4.0) and sea sand, and then ground. RNA was subsequently extracted with phenol/chloroform/iso-amylalcohol and digested with RNAse-free DNAse . The concentration and purity of RNA was determined spectrophotometrically at 260 and 280 nm. A 2-μg portion of RNA was used for reverse transcription with M-MLV reverse transcriptase (Promega). PCR amplification of target genes was performed with GoTaq© DNA polymerase (Promega), and products were separated on 1.5% agarose gels. A complete list of all primers and gene identifiers for the target genes is shown in Leaves from 2-week-old Arabidopsis plants were frozen in liquid nitrogen and 100 mg of leaf material was processed per sample. RT-PCR was carried out as described in detail by http://www.cellsignal.com).Total root protein extracts were prepared using the phenol protein extraction method, according to http://www.thermofisher.com) linear ion trap mass spectrometer, and MS/MS spectra were interpreted in Mascot 2.2  and Bioworks 3.3 (Thermo). The database used for the search was the TAIR9 protein database (ftp://ftp.arabidopsis.org/home/tair/Sequences/blast_datasets/TAIR9_blastsets/TAIR9). Further details on protein identification can be found in S]-X-[KRP], [RK]-X-X-[ST]-X-[KRP], [KR]-X-X-[ST], ϕ-X-[KR]-X-X-S-X-[KRP], or [RKHYCDE]-X-X-[KR]-X-X-S-X-X-[KR], see P-score of a protein X of length l as:Coomassie-stained gel spots were excised from the 2D gel and used for nano-electrospray LC-MS/MS investigations after a destaining tryptic digest. After separation on a C18 reverse phase column, mass spectra were obtained in an LTQ (Thermo, i occurs ai times in a given protein X, with the probability mi to occur by chance in the total Arabidopsis proteome.Where the motif


Myxoid liposarcoma is a relatively common malignant soft tissue tumor, characterized by a  translocation resulting in a FUS-DDIT3 fusion gene playing a pivotal role in its tumorigenesis. Treatment options in patients with inoperable or metastatic myxoid liposarcoma are relatively poor though being developed and new hope is growing.Using kinome profiling and subsequent pathway analysis in two cell lines and four primary cultures of myxoid liposarcomas, all of which demonstrated a FUS-DDIT3 fusion gene including one new fusion type, we aimed at identifying new molecular targets for systemic treatment. Protein phosphorylation by activated kinases was verified by Western Blot and cell viability was measured before and after treatment of the myxoid liposarcoma cells with kinase inhibitors. We found kinases associated with the atypical nuclear factor-kappaB and Src pathways to be the most active in myxoid liposarcoma. Inhibition of Src by the small molecule tyrosine kinase inhibitor dasatinib showed only a mild effect on cell viability of myxoid liposarcoma cells. In contrast, inhibition of the nuclear factor-kappaB pathway, which is regulated by the FUS-DDIT3 fusion product, in myxoid liposarcoma cells using casein kinase 2 inhibitor 4,5,6,7-tetrabromobenzotriazole (TBB) showed a significant decrease in cell viability, decreased phosphorylation of nuclear factor-kappaB pathway proteins, and caspase 3 mediated apoptosis. Combination of dasatinib and TBB showed an enhanced effect.in vitro and inhibition of nuclear factor-kappaB pathway activation by inhibiting casein kinase 2 using TBB, of which the effect is enhanced by Src inhibition using dasatinib, offers new potential therapeutic strategies for myxoid liposarcoma patients with advanced disease.Kinases associated with activation of the atypical nuclear factor-kappaB and the Src pathways are the most active in myxoid liposarcoma  Ecteinascidia turbinate. By binding to the DNA minor groove, ET-743 forms covalent adducts with the N2-position of guanine through its carbinolamine moiety. As a result, the minor groove bends toward the major groove. The cytotoxic activity of ET-743 is largely based on its interaction with nucleoside excision repair machinery, as well as through the induction of double strand breaks[Myxoid liposarcoma accounts for 40% of all liposarcomas and occurs most commonly in the extremities . In abound breaks-11. Phasnd breaks,13. Durind breaks. Notablend breaks. This nend breaks,17.Data on activated pathways in myxoid liposarcoma are sparse,19. By uGU933437). COBRA-FISH of both myxoid liposarcoma cell lines showed the myxoid liposarcoma specific t translocation. The precise karyotype of 402-91 was: 46, X, der(Y)t, t, der(8)t[FISH of the primary myxoid liposaromas showed the tumor specific t12;16) in three out of four cases  expressed comparable amounts of total Src and NF-kappaB p65. Phosphorylation of Src (Y419) was present in all samples figure  confirmiWST-1 analysis of GIST882 showed a profound decrease in cell viability of up to ~ 80% relative to the DMSO control at even low dosages of Src-inhibitor dasatinib figure . The decTo investigate the effect of dasatinib on Src signalling, a good responsive  was found figure . At a doin vitro and performed subsequent pathway analysis.Treatment options for myxoid liposarcoma patients with advanced disease are poor. Recently, the chemotherapeutic drug Trabectedin showed promising results in phase I and II trials in advanced disease though adverse effects have also been reported,25. SmalWe previously established the reliability of kinome profiling using Pepchip in untreated versus imatinib treated GIST882 cell line which correctly identified the pathways known to be involved in GIST. Moreovevitro and in vivo with peroxisome proliferator-activated receptors gamma agonists induced terminal differentiation[We could demonstrate activation of the peroxisome proliferator-activated receptor gamma pathway, which could be expected since it has been shown to play a pivotal role in adipocytic differentiation and is regulated by the FUS-DDIT3 fusion product-28Figur. The DDIentiation, althougentiation. Until tentiation. We descentiation,35.et al. has recently shown that NF-kappaB is a major factor controlling IL8 transcription in FUS-DDIT3 expressing cells. This could be explained by direct binding of FUS/DDIT3 to the C/EBP-NF-kappaB composite site of the immediate promoter region of IL8. Moreover, FUS/DDIT3-GFP expressing cell lines showed upregulation of the NF-kappaB controlled genes LCN2 and MMP1 whereas DDIT3 had little effect. These findings were also quantitatively confirmed by RT-PCR[We showed that kinases associated with NF-kappaB pathway were highly active in myxoid liposarcoma. In the atypical (IKK independent) NF-kappaB pathway, phosphorylation of inhibitors of NF-kappaB (IkB), and subsequent activation of NF-kappaB p65) is controlled by casein kinase 2 and tyrosine kinase-dependent pathways , whereas viability was only 5% at lower concentration of dasatinib (500 nM) at IC50 for TBB (figure Combinations of different drugs (including dasatinib) have been shown to act synergistically in many tumors and combination drug therapy is commonly used in cancer treatment. Recentlin vitro, the effect of which can be enhanced by inhibiting src- signalling using dasatinib.In conclusion our results indicate that the NF-kappaB and Src pathway include the most active kinases in myxoid liposarcoma, and inhibition of casein kinase 2 and thereby interference with kinases associated with the NF-kappaB pathway decreases cell viability Dasatinib  was obtained from Bristol-Myers Squibb  and TBB from Calbiochem . Both drugs were dissolved in Dimethylsulfoxide (DMSO).The two myxoid liposarcoma cell lines 402-91 and 1765-92, and gastro-intestinal stroma cell tumor cell line (GIST882) were kindly provided by Prof. Dr. P. Aman  and Prof. Dr. J. Fletcher  respectively,54. JurkFUS-forward, CAG AGC TCC CAA TCG TCT TAC GG and DDIT3-reverse, GAG AAA GGC AAT GAC TCA GCT GCC.Diagnosis of the primary tumors from which the cultures were obtained was performed on histology. Primary tumors were analyzed for their tumor specific translocation with double-fusion fluorescence in situ hybridization (FISH) and cell lines were karyotyped with Combined Binary Ratio Labeling (COBRA) as previously described -57. In phttp://www.pepscanpresto.com/index.php?id=30. Cells were harvested during their exponential growth phase and lysated as previously described. Concentration of the protein lysates was measured using the DC Protein Assay . Analysis was performed as described earlier, including the two serum-starved samples[6 hits were collected.Kinase substrate peptide arrays  containing 1024 different kinase substrates spotted in triplicate together with 16 negative, and 16 positive controls were used and successfully used in prior studies . The dishttp://www.bioconductor.org. Quality of the triplicates and distribution of the data was assessed and quartile normalization (Affyio) was performed as previously described[http://www.ingenuity.com). IPA is a literature based program that calculates the probability of involvement of identifiers, in this case combinations of kinases, in 74 different pathways. Data of the myxoid liposarcoma cell lines and cultures were averaged to find the common denominators that are active in all cultures[The scanned images were analyzed and quantified using ImageQuant software . For further data mining R-packages Affyio and Limma were used described. Median  cultures. To ensu cultures.Western blotting was performed as previously described. Rabbit Measurement of metabolic activity by a WST-1 colorimetric assay  was used as a read-out system for cell viability in response to kinase inhibitors. Dasatinib was used to inhibit Src-pathway; TBB was used to inhibit casein kinase 2, which is an important kinase in atypical NF-kappaB signalling. After harvesting, 2000 cells/well of every cell line and primary culture were seeded into 96-well flat-bottom plates. After 24 hours, increasing concentrations of the drugs  were added or 0,1% DMSO as vehicle control, each condition in quadruplicate. Ten percent serum supplementation was used for all experiments. After 3 days of treatment, absorbance was measured on a Victor Multilabel Counter 1420-042  at 450 nm, and was corrected for background and averaged. GIST882 and Jurkat cell lines were used as positive controls for dasatinib and TBB experiments, respectivelyy. In comThe authors declare that they have no competing interests.in vitro studies and DNA sequencing and drafted the manuscript. YMS participated in design of the kinome assay, immunoblotting and statistical analysis. IHB participated in kinome studies and immunoblotting. KS carried out the karyotyping, analysis of DNA sequences and participated in design of cell culturing. PCWH participated in design and coordination and helped to draft the manuscript. JVMGB designed and supervised the study and helped to draft the manuscript. All authors read and approved the final manuscript.SMW carried out kinome studies, immunoblotting, Top 100 of activated kinases in MSCs obtained by kinome analysis. Table shows list of kinases found to be activated in mesenchymal stem cells, in decreasing order. The intensity correlates with the radioactivity of 33P incorporated in the substrates by their active kinases. The number of hits are the number of substrates related to the respective kinase.Click here for file


These statins  may exert other effects besides from their cholesterol-lowering actions, including inhibition of platelet activation. Platelet activation is relevant to a variety of coronary heart diseases. Although the inhibitory effect of simvastatin in platelet activation has been studied; the detailed signal transductions by which simvastatin inhibit platelet activation has not yet been completely resolved.The aim of this study was to systematically examine the detailed mechanisms of simvastatin in preventing platelet activation. Platelet aggregation, flow cytometric analysis, immunoblotting, and electron spin resonance studies were used to assess the antiplatelet activity of simvastatin.2+]i mobilization, thromboxane A2 (TxA2) formation, and phospholipase C (PLC)γ2, protein kinase C (PKC), and mitogen-activated protein kinases  phosphorylation in washed platelets. Simvastatin obviously increased both cyclic AMP and cyclic GMP levels. Simvastatin markedly increased NO release, vasodilator-stimulated phosphoprotein (VASP) phosphorylation, and endothelial nitric oxide synthase (eNOS) expression. SQ22536, an inhibitor of adenylate cyclase, markedly reversed the simvastatin-mediated inhibitory effects on platelet aggregation, PLCγ2 and p38 MAPK phosphorylation, and simvastatin-mediated stimulatory effects on VASP and eNOS phosphorylation.Simvastatin (20-50 μM) exhibited more-potent activity of inhibiting platelet aggregation stimulated by collagen than other agonists . Simvastatin inhibited collagen-stimulated platelet activation accompanied by [Ca2 cascade, and finally inhibition of platelet aggregation.The most important findings of this study demonstrate for the first time that inhibitory effect of simvastatin in platelet activation may involve activation of the cyclic AMP-eNOS/NO-cyclic GMP pathway, resulting in inhibition of the PLCγ2-PKC-p38 MAPK-TxA A high incidence of atherosclerosis and thrombotic complications has been associated with hypercholesterolemia. Blood cholesterol levels are of fundamental importance in the pathogenesis of coronary artery diseases (CAD). Elevations of low-density lipoprotein (LDL) levels are not only linked to an increased risk for atherosclerosis but may also exert prothrombotic effects via platelet activation . The eff2 formation or changing cholesterol content on platelet membrane by statins has been reported i rise was measured using a fluorescence spectrophotometer  with excitation wavelengths of 340 and 380 nm, and an emission wavelength of 500 nm  [The ESR method used a Bruker EMX ESR spectrometer as described previously . In brieontrol)] .P < 0.05 was considered statistically significant.The experimental results are expressed as the means ± S.E.M. and are accompanied by the number of observations. The experiments were assessed by the method of analysis of variance (ANOVA). If this analysis indicated significant differences among group means, then each group was compared using the Newman-Keuls method. 50 value of simvastatin for platelet aggregation induced by collagen was approximately 30 μM. The solvent control (0.5% DMSO) did not significantly affect platelet aggregation stimulated by agonists in washed platelets  exhibited potent activity of inhibiting platelet aggregation and the ATP-release reaction stimulated by collagen . It also significantly inhibited platelet aggregation stimulated by thrombin , AA  or U46619 , a prostaglandin endoperoxide at higher concentrations (70-100 μM) Fig.  and 1B. ets Fig. . When plTrimeresurus flavoviridis snake venom i, and this increase was markedly inhibited in the presence of simvastatin .Free cytoplasmic Ca2 compared to collagen-activated platelets. Simvastatin (30 and 50 μM) concentration-dependently inhibited TxB2 formation in platelets stimulated by collagen (1 μg/ml). PLC hydrolyzes phosphatidylinositol 4,5-bisphosphate (PIP2) to generate two secondary messengers: inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG) [As shown in Figure ol (DAG) . DAG actTo further investigate the inhibitory mechanisms of simvastatin in platelet activation stimulated by collagen, we further detected MAPK signaling molecules including p38 MAPK, JNKs, and ERKs. The immunoblotting analysis revealed that simvastatin (50 μM) inhibited p38 MAPK Fig.  and JNKs1 (10 μM) markedly increased approximately 4.3-fold of cyclic AMP level compared with the resting group     and simvastatin (30 and 50 μM) markedly induced VASP Ser157 phosphorylation  and simvastatin (50 μM)  Fig. . We also 3) Fig. . The add 3) Fig. . NO was  3) Fig. . It was latelets . In thision Fig. . SQ22536μM) Fig. . FurtherμM) Fig. . On the μM) Fig.  and 4F. 1 (10 μM) and simvastatin (50 μM)  formation was induced in collagen (1 μg/ml)-activated platelets compared to resting platelets  phosphorylation was markedly activated by both PGEμM) Fig. . The simμM) Fig. . On the ets Fig.  and 5b; ets Fig.  and 5d. in vitro are reasonable higher than that of blood concentrations obtained during a simvastatin regimen in vivo. However, the concentration employed is closely to that of other in vitro studies (20-80 μM) [This study demonstrates for the first time that simvastatin inhibits platelet activation via a novel pathway: activation of cyclic AMP-eNOS/NO-cyclic GMP and inhibition of MAPK phosphorylation  in washed platelets. Simvastatin exhibited more-potent activity at inhibiting collagen-induced platelet aggregation than other agonists. For the clinical therapy, the approved starting dose of simvastatin for most patients is 20 mg, and the maximal dose is 80 mg. In this study, the concentrations of simvastatin were employed at 30 and 50 μM, and the concentration of collagen was used at 1 μg/ml to trigger platelet aggregation. In general, concentrations of collagen were employed for platelet aggregation of from 0.1 to 5 μg/ml. In an attempt to elucidate the detailed mechanisms of pharmacological interest, we used a higher concentration (1 μg/ml) of collagen to induce a more-pronounced signal transduction in platelets . Therefore, the pharmacological concentrations (30-50 μM) of simvastatin employed to inhibit platelet aggregation 0-80 μM) ,14,15.2), resulting in the production of the second messengers, inositol 1,4,5-trisphosphate (IP3) and DAG [2 formation, a stable metabolite of TxA2, was markedly inhibited by simvastatin. TxA2 is important for collagen-induced platelet aggregation. This may explain the more-potent activity of simvastatin in inhibiting collagen-induced platelet aggregation than other agonists (thrombin and U46619).Stimulation of platelets by agonists  causes marked alterations in phospholipid metabolism. The activation of PLC results in the degradation of phosphoinositides, notably, phosphatidylinositol 4,5-bisphosphate . p38 MAPK is essential for the stimulation of cPLA2, which catalyzes AA release to produce TxA2 [2-dependent platelet aggregation pathway. Simvastatin significantly inhibits TxA2 formation, at least in part, via inhibition of p38 MAPK phosphorylation.MAPKs consist of three major subgroups. Growth factors preferentially activate ERKs (p44 ERK1 and p42 ERK2), which are involved in proliferation, adhesion, and cell progression , whereastivation . On the tivation . Among tuce TxA2 ; thus, p2+ concentrations by the uptake of Ca2+ into the dense tubular system (DTS) which negatively affects the action of PLC and/or PKC [Activation of human platelets is inhibited by two intracellular pathways regulated by either cyclic AMP or cyclic GMP. The importance of cyclic AMP and cyclic GMP in modulating platelet reactivity is well established . In addid/or PKC . Therefod/or PKC . Most ced/or PKC . Simvastd/or PKC . In thisd/or PKC . In thisd/or PKC .2+ during the initial phase of platelet activation processes, and PKC is involved in receptor-mediated free radical production in platelets [Reactive oxygen species  derived from platelet activation might amplify platelet reactivity during thrombus formation. Free radical species act as secondary messengers that increase cytosolic Calatelets . The ant2 cascade, thereby leading to inhibition of platelet aggregation. Hypercholesterolemic patients usually associate with a high incidence of atherosclerosis and thrombotic complications. This study provides a new insight of antiplatelet mechanisms of simvastatin to explain its clinical protective effect in CAD.In conclusion, the most important findings of this study demonstrate for the first time that the antiplatelet activity of simvastatin may involve an increase of the cyclic AMP-eNOS/NO-cyclic GMP pathway, followed by inhibition of the PLCγ2-PKC-p38 MAPK-TxAThe authors declare that they have no competing interests.YML and WFC carried out the platelet aggregation study and drafted the manuscript. DSC carried out the ESR study. TJ, SYH, and JJL carried out the immunoblotting study. GH performed the statistical analysis. JRS conceived of the study, and participated in the design and coordination, and collectively prepared the manuscript. All authors read and approved the final manuscript.


B. anthracis spores of the plasmidless dSterne strain in comparison with the Sterne strain containing the toxigenic plasmid (pXO1). The differential regulation of phosphorylation was found in the mitogen-activated protein kinase cascade , the PI3K cascade , and downstream in the case of the proapoptotic BAD and the transcription factor STAT3. Both strains stimulate phosphorylation of CREB and inhibit phosphorylation of 4E-BP1 required for activation of cap-dependent translation. Downregulation of the survival AKT phosphorylation by the Sterne strain inhibits the process of Ca2+-dependent homophilic interaction of E-cadherin (EC) upon formation or repair of cell–cell contacts. Both lethal and edema toxins produced by the Sterne strain inhibit the AKT phosphorylation induced during the EC-mediated signaling. Activity of ERK1/2 and p38 inhibitors indicates that inhibition of AKT phosphorylation takes place through the ERK1/2-PI3K crosstalk. In Sterne spore-challenged mice, a specific inhibitor of PI3K/AKT, wortmannin, accelerates the lethal outcome, and reduction of AKT phosphorylation in the circulating blood cells coincides with the death of animals. We conclude that the PI3K/AKT pathway controlling the integrity of epithelium plays an important survival role in anthrax infection.The effect of anthrax infection on phosphoprotein signaling was studied in human small airway lung epithelial cells exposed to  Bacillus anthracis spores. The virulence of B. anthracis is mainly attributed to its lethal and edema toxins  encoded by the XO1 plasmid and the antiphagocytic capsule encoded by the XO2 plasmid. LeTx is a proteolytic inhibitor of mitogen-activated protein kinases (MAPKKs), while EdTx is a bacterial adenylate cyclase generating increased levels of intracellular cyclic AMP (cAMP)  while taking into account the critical role of lung function in the outcome of inhalation anthrax  . SeveralAKT (protein kinase B) is an established pluripotent key mediator of cell survival after various insults, including growth factor withdrawal, chemical and physical stress, treatment with therapeutic agents, and ischemic shock . Class 1B. anthracis infection on host cell phosphoprotein signaling in infected HSAECs, including inhibition of the PI3K/AKT pathway. We also show that this pathway is causally important for the survival of B. anthracis spore-challenged mice. LeTx and EdTx contribute to the inhibition of AKT phosphorylation and thus interfere with the signaling required for the assembly of the EC-mediated adherens junctions.In this report we demonstrate a broad impact of Cell culture reagents were from CellGro . Antibodies against total and phosphorylated forms of the following proteins used for reverse-phase protein microarrays (RPMA) and Western blots were from Cell Signaling Technology  and were used at the dilutions indicated: 1 : 20 for p70 S6 kinase (Thr389); 1 : 50 for c-Abl (Thr 735), Stat5 (Tyr694), 4E-BP1 (Ser65); 1 : 100 for AKT (Ser473), MEK1/2 (Ser 217/221), pIKBa (Ser32/Ser36), Bad , 4E-BP1 (Thr70), GSK-3α/β (Ser21/9), CREB (Ser 133), Stat3 , Jak1 (Tyr1022/1023), FAK (Tyr576/577), Etk (Tyr 40), Elk-1 (Ser383), MARCKS (Ser152/156); 1 : 200 for mTOR (Ser2448), eNOS (Ser1177), Pyk2 (Tyr402), FADD (Ser194), Stat6 (Tyr641), Bcl-2 (Ser70); 1 : 250 for p38 (Thr180/Tyr182), IL-1β-cleaved (Asp116); 1 : 400 for p90RSK (Ser380); 1 : 500 for PKC-δ (Thr505), PKC-α/β (Thr638/641), PKC-θ (Thr538), caspase-7 cleaved (Asp198), caspase-9 cleaved (Asp330), caspase-3 cleaved (Asp175), ERK 1/2 (Thr202/Tyr204), pPKC-z (Thr410/403), Src (Tyr527), Stat1 (Tyr701), Bax; 1 : 1000 for actin, 4E-BP1 (Thr37/46), EC, Bcl-xL; 1 : 2000 for eIF4G (Ser1108). Recombinant protective antigen, lethal factor, and edema factor were from List Biological Laboratories . Other reagents were from Sigma-Aldrich .2. The cells were adapted to these culture conditions during four passages and then were used for the preparation of the frozen stock. Further experiments were performed with cells at passages between 5 and 10. For microarray experiments, confluent HSAECs from one of the donors (106 per well in 12-well plates) were incubated in Complete Serum-Free Medium® (CellGro) plus 1% FCS for 16 h before challenge with B. anthracis spores at the multiplicity of infection (MOI) 1 and 10. According to the manufacturer this medium is a proprietary serum-free and low-protein formulation based on DMEM/F12, RPMI 1640, and McCoy's 5A that does not contain any insulin, transferrin, cholesterol, growth or attachment factors. A mixture of trace elements and high molecular weight carbohydrates, extra vitamins, a nonanimal protein source, and a small amount of high quality bovine serum albumin (1 g L−1) are added to support growth and viability over long-term passages of hybridomas, suspension, and adherent cultures. The toxigenic B. anthracis Sterne strain 43F2  and the nontoxigenic, plasmidless strain dSterne  were from Colorado Serum Company (CO) and the National Center for Biodefense and Infectious Diseases, George Mason University (VA), correspondingly. The absence of pXO1 in the dSterne strain was confirmed by PCR with the protective antigen-specific primers. Both strains have identical growth curves in the culture medium used for challenge experiments  from two different donors were grown according to the vendor's protocol in Ham's F12 medium supplemented with nonessential amino acids, pyruvate, β-mercaptoethanol and 10% fetal calf serum (FCS) at 37 °C in an atmosphere of 5% CO−1 of each penicillin and streptomycin) added to the samples 1 h before assay to inhibit the metabolic influence of bacterial vegetative cells. Fluorescence of the dye was measured at 590 nm after excitation at 530 nm. In the negative control experiments, bacteria grown without HSAECs did not demonstrate Alamar Blue fluorescence after incubation with antibiotics.The viability of cells after exposure to spores was determined using the Alamar Blue (Sigma-Aldrich). Alamar Blue is a water-soluble, nontoxic, fluorometric/colorimetric growth indicator. Cellular growth and metabolism reduce the dye and cause a color change to red. The cells grown in 96-well plates were incubated with Alamar Blue in the presence of antibiotics  according to manufacturer's protocol using fluorescence microscope Nikon Eclipse 90i with C1SHS camera. Briefly, after incubation with SUPs, cells grown on slides were washed 3 times with the binding buffer. Annexin V conjugated with Cyt3.18 dye was added for 10 min, the cells were washed five times with the binding buffer, and treated with fixation/permeabilization solution  for 20 min. Finally, the slides were washed three times with phosphate-buffered saline (PBS), dried at room temperature, and mounted using antifade reagent Prolong Gold (Invitrogen) containing a nuclear DAPI stain.In the experiments with bacterial culture supernatants (SUPs), confluent HSAECs were seeded into the 12- or 48-well culture plates, cultivated as described above, washed three times with Hanks balanced salt solution (HBSS), and incubated with SUPs at 37 °C, 5% CO4 per well of eight-chamber plate from BD Biosciences), grown in 0.5 mL well−1 of Ham's F12 medium with 10% FCS at 37 °C and 5% CO2 for 7 days, and treated with SUPs of Sterne and dSterne strains for 4 h. After treatment the cells were briefly washed with Complete Serum-Free Medium®, fixed with 3.2% paraformaldehyde in HBSS with Ca2+ and Mg2+ for 10 min, washed twice with the blocking buffer , permeabilized with cold 1% Triton X-100 in the blocking buffer for 10 min, washed three times with the blocking buffer, and finally incubated in the blocking buffer for 30 min. The cells were then incubated with the first antibody  in blocking buffer (2 μg mL−1) at 4 °C overnight, washed three times in the washing buffer , incubated with 2 μg mL−1 of secondary antibody in the blocking buffer  for 30 min, washed three times in the washing buffer, and finally mounted as described above.For immunofluorescent staining with anti-EC antibody, HSAECs were seeded  loading buffer with cocktail of protease inhibitors (Pierce), phosphatase inhibitors , 2 mM EDTA, and 6 mM dithiothreitol.alphaease fc (Alpha Innotech). For each antibody, the average pixel intensity value for negative control (staining with only second antibody) was subtracted from the average pixel intensity value for specific antibody and then divided by the corresponding value of the Sypro-stained total protein slide. For RPMA, each sample dilution curve in each experiment was fitted with a nonlinear approximation equation and statistically evaluated using the software prizm . The dilutions of different samples, which resulted in the same signal intensity arbitrarily chosen within the linear response interval, were used to calculate relative analyte concentrations and their confidence intervals (P=0.05). The microarray experiments were repeated to ensure consistent results. HSAECs from two different donors demonstrated similar results (not shown). Signaling proteins demonstrating difference between control and challenged cells were further tested with Western blots using 20 μL of cell lysates, 1 : 1000-diluted primary antibodies, and 1 : 7500-diluted secondary antibody. Proteins were transferred to nitrocellulose membrane using iBlot Gel Transfer Device (Invitrogen). The membranes were developed with SuperSignal West Femto Maximum Sensitivity Substrate (Pierce), and band intensities were measured with a Molecular Imager ChemiDoc XRS System . The intensities of bands were calculated relative to untreated controls after densitometry using the quantity one 4.6.5 program (Bio-Rad). All measurements were performed in triplicate.Three nanoliters of each sample were arrayed by direct contact printing using a high-resolution 2470 arrayer  onto nitrocellulose slides . Samples were printed as duplicates of the five-point serial dilution curves to ensure the linear detection range for the antibody concentrations used. Slides were kept at −20 °C before analysis with antibodies. To estimate the total protein amount, selected slides were stained with Sypro Ruby Protein Blot Stain  and visualized on a Fluorchem imaging system . Slides were stained with specific antibodies on an automated slide stainer  using a biotin-linked peroxidase-catalyzed signal amplification. The arrayed slides were placed into 1 × Re-Blot solution  for 15 min, washed two times for 5 min each in PBS, placed into I-Block solution  in PBS/0.1% Tween-20 for at least 2 h, and then immunostained using an automatic slide stainer  using manufacturer-supplied reagents. Briefly, the slides were incubated for 5 min with hydrogen peroxide, rinsed with high-salt Tris-buffered saline  supplemented with 0.1% Tween-20, blocked with avidin block solution for 10 min, rinsed with CSA buffer, and then incubated with biotin block solution for 10 min. After another CSA buffer rinse, 5-min incubation with protein block solution was followed by air drying. The slides were then incubated with either a specific primary antibody diluted in Dako Antibody Diluent or, as a control, with only DAKO Antibody Diluent for 30 min. Before use on the RPMA, every antibody underwent extensive validation for specificity . The slides were then washed with CSA buffer and incubated with a secondary biotinylated goat anti-rabbit immunoglobulin G H+L antibody (1 : 5000)  for 15 min. For amplification purposes, the slides were washed with CSA buffer and incubated with streptavidin–horseradish peroxidase for 15 min, followed by a CSA buffer rinse. Slides were then incubated in diaminobenzidine chromogen diluted in Dako diaminobenzidine diluent for 5 min, washed in deionized water and imaged using a UMAX PowerLook III scanner  at 600 dpi. The images were analyzed with software ad libitum and were challenged with B. anthracis Sterne spores  on day 0. Survival of animals was monitored for 10 days. Wortmannin (Sigma-Aldrich) was administered i.p. 1 h before the spore challenge, and then once daily until death. To obtain blood samples, the challenged animals were bled from the orbital sinus vein and then immediately sacrificed by cervical dislocation. Blood was mixed with equal volume of 2 × SDS-PAGE loading buffer containing 12 mM dithiothreitol, 2 × cocktail of protease inhibitors (Pierce), phosphatase inhibitors , and 4 mM EDTA. Samples were boiled for 10 min and used for Western blot with pAKT-specific antibody as described above. Densitometry was performed and relative intensities of bands were calculated. Analysis was carried out in triplicate. The protocol of experiments was approved by the Institutional Animal Care and Use Committee of George Mason University, Manassas, VA.Male 6–8-week-old DBA/2 mice (Jackson Labs) received food and water All data are presented as mean±confidence interval with a significance level of 0.95 calculated using the SDs of the measurements. The Kaplan–Meier log-rank statistical test was applied to evaluate reliability of survival data.B. anthracis infection, we used a model of HSAECs challenged with B. anthracis spores, which germinated and grew as vegetative bacteria atop of the host cells. We suggested that this model would be instructive regarding the initial interaction of lung epithelium with inhaled spores, as well as the late septicemic stage of infection characterized by dissemination of vegetative bacteria and their secreted products to the lungs and other epithelium-reach organs. Cell lysates were prepared at different times postchallenge to encompass the period of infection from 10 min to 8 h at MOI 1 and 10. For initial identification of signaling proteins the lysates were used for fabrication of RPMA on nitrocellulose membrane slides. Each slide was then probed with one of 40 different antibodies specific against phosphorylated forms of signaling proteins  selected to monitor the molecular networks involved in host responses most likely affected by bacterial exposure, namely survival, apoptosis, inflammation, growth, differentiation, and immune response. This microarray technique was extensively validated in our previous studies (P<0.05) difference from those in the control untreated cells at one or several time points upon exposure to either toxigenic or nontoxigenic B. anthracis strain . All proteins whose phosphorylations were significantly changed during the infection in HSAECs were found to belong to a single direct-interaction network (P<10−5) where all signaling nodes are interconnected, and no irrelevant nodes without direct connections are present. The high functional importance of these network proteins is reflected in the connectivity index measuring the number of interactions with other proteins in the database . Among the key host cell signaling proteins whose phosphorylation was differentially inhibited by the toxigenic Sterne strain and stimulated by the dSterne strain are the stress-activated kinase p38, ERK1/2 (p44/42 MAPK), and its downstream target, p90 RSK, all members of the prosurvival MAPKs pathway. Consistent with these observations, MAPKKs 1/2 and 3/6 regulating the downstream kinases ERK and p38 are well-known specific targets of LeTx proteolytic activity . In addiB. anthracis infection on host cell signaling could be mimicked by the soluble factors secreted by bacterial cells. For this purpose, we grew B. anthracis cultures in the conditions used for the RPMA experiments and tested the supernatants of these cultures (SUPs), as well as the pelleted and washed bacteria harvested at different stages of growth  for their effects on cell viability, induction of apoptosis, and inhibition of AKT phosphorylation.In order to reduce the complexity of our experimental model, we tested whether the effect of Freshly confluent HSAECs incubated with SUPs for 2 h demonstrated no detectable loss of HSAECs' viability compared with control cells in the assays with Alamar Blue  and carb−1 of both penicillin and streptomycin to prevent bacterial growth) did not affect AKT phosphorylation (not shown). In order to explore the mechanism of differential AKT phosphorylation, we suggested that it might be relevant to changes in the intercellular contacts known as sites of intense phospho-tyrosine signaling  disrupts this interaction and causes internalization of EC followed by its ubiquitination and degradation  phosphorylation was detected using the 8-h SUPs after 2-h incubation . The was of pAKT . HoweverB. anthracis proteins, including LeTx, changed a pattern of EC staining at the cell–cell contacts of cultured murine mammary gland epithelial cells . The HSAECs were pretreated with purified LeTx and EdTx for 1 h and tested in the calcium switch experiments. Both toxins individually and in combination with each other were capable of inhibiting AKT phosphorylation to about 20% of its Ca2+-induced level in control untreated cells, without a detectable change in the amount of cell-associated EC  in the SUP at 4 h after spore inoculation in the amount of 1 μg mLiated EC .In order to confirm that the effect of LeTx was relevant to MAPKK inactivation, we carried out experiments with chemical inhibitors of MAPKKs. In the conditions of calcium switch, PD98059, a specific inhibitor of ERK1/2, reduced AKT phosphorylation similar to LeTx . This isGeneration of cAMP is the only known function of EdTx. To confirm the existence of a cAMP-AKT pathway, the cells were treated with forskolin, an inducer of cellular adenylate cyclase, in the presence or absence of the phosphodiesterase inhibitor IBMX (which prevents intracellular degradation of cAMP). The level of pAKT after stimulation with serum in culture medium was considerably reduced by this treatment . OverallB. anthracis infection and extend our analysis beyond pure correlative observations, mice were administered an inhibitor of class I PI3K, wortmannin, and then challenged with spores of the toxigenic Sterne strain. (The nontoxigenic strain does not produce a productive infection and therefore could not be tested.) Similar approaches have been used previously to investigate the role of the PI3K pathway in endotoxemic and oxidant-injured mice (B. anthracis might contribute to B. anthracis' overall virulence.In order to test the relevance of AKT inhibition to the virulence of B. anthracis strains detected in HSAECs takes place in vivo during systemic infection, we challenged mice with a lethal dose of Sterne spores and analyzed the levels of AKT activation in circulating blood cells from mice bled at different stages of infection. In the control group, animals were challenged with the same amount of nonlethal dSterne spores. To determine whether differential inhibition of AKT signaling by the two B. anthracis using an RPMA microarray to broadly measure cell signaling pathway activation and confirmed these findings using Western blots. Although LeTx is known to inhibit the MAPK cascade, the full picture of host cell signaling changes in response to anthrax infection remains largely unknown. The ability to simultaneously detect changes in the phosphorylation of a large number of proteins over a time course in the comparative experiments with the matched pair of isogenic pXO1-positive and pXO1-negative B. anthracis strains helped us expand the information available regarding specific signaling pathways altered by the infection. Our results showed that HSAECs responded to the nonlethal B. anthracis strain with a cascade of host signaling emanating from major prosurvival MAPK and PI3K/AKT pathways, while infection with the lethal strain caused a reduction in activation/phosphorylation of key members of entire cell survival signaling networks. A number of previous studies did not utilize this comparative approach and typically reported results using only virulent strains or toxins (Lung epithelial cells are positioned at the foremost interface between the environment and the internal compartments of the body and provide both barrier and signaling functions to protect against infection by airborne pathogens. Therefore, these cells are expected to behave as a robust but sensitive barrier that quickly responds to pathogenic insults. In this report, we characterized the early innate responses of primary lung epithelial cells to the pathogenic factors secreted by r toxins . ConsequThe fundamental importance of MAPK and AKT in cell behavior led us to explore the correlation between host lung epithelial cell signaling and the most important direct biological outcome of anthrax exposure, mortality. Reduced phosphorylation of AKT, due to its central role in survival, typically follows proapoptotic insults, while the constitutive activation of AKT is able to block cell death induced by a variety of apoptotic stimuli. Both animal and recent patient studies have established lung endothelial and epithelial apoptosis as an important part of the pathogenesis of acute lung injury (B. anthracis into culture medium through a mechanism, which controls the calcium-dependent interaction of EC upon initiation of cell–cell contacts; this interaction normally induces a transient increase in AKT phosphorylation followed by the maturation of adherens junctions. Therefore, anthrax toxins are expected to interfere with the processes of epithelial development and repair after injury (We found that reduced AKT phosphorylation in HSAECs was caused mainly by anthrax toxins secreted by r injury . The resAKT activates survival-related gene expression by transcription factors NF-κB and CREB and by apoptosis regulatory molecules, including BAD and mTOR pathway components (BAD phosphorylation takes place through the same major pathways as CREB (AKT is able to directly phosphorylate a constitutively expressed regulator of cellular glucose metabolism, GSK-3β. Inhibition of GSK-3β by AKT-mediated phosphorylation takes place in response to different external prosurvival stimuli . We founThe optimal activation of the PI3K and the MAPK pathways depends on JAK/STAT signaling following binding of cytokine/interferon receptors with their ligands (B. anthracis gene knockouts in order to determine more precisely the bacterial factors involved in the host signaling response.We conclude that interference with the host cell PI3K/AKT response represents an important part of a multifaceted anthrax pathogenic strategy. Kinomic analysis of host cell signaling provides a new opportunity to dissect host–pathogen interactions for possible therapeutic intervention candidates and mechanistic elucidation. Future studies will include experiments using specific


However, to our surprise we found that activation of PAR2 by trypsin or the specific activating peptide SLIGKV-OH strongly inhibited both the phosphorylation and activity of JNK. In contrast neither p38 MAP kinase nor ERK activation was affected although TNFα stimulated IκBα loss was partially reversed. The inhibitory effect was not observed in parental cells nor in cells expressing PAR4, however inhibition was reversed by pre-incubation with the novel PAR2 antagonist K14585, suggesting that the effect is specific for PAR2 activation. SLIGKV-OH was found to be more potent in inhibiting TNFα-induced JNK activation than in stimulating JNK alone, suggesting agonist-directed signalling. The PKC activator PMA, also mimicked the inhibitory effect of SLIGKV-OH, and the effects of both agents were reversed by pre-treatment with the PKC inhibitor, GF109203X. Furthermore, incubation with the novel Gq/11 inhibitor YM25480 also reversed PAR2 mediated inhibition. Activation of PAR2 was found to disrupt TNFR1 binding to RIP and TRADD and this was reversed by both GF109203X and YM25480. A similar mode of inhibition observed in HUVECs through PAR2 or P2Y2 receptors demonstrates the potential of a novel paradigm for GPCRs linked to Gq/11, in mediating inhibition of TNFα-stimulated JNK activation. This has important implications in assessing the role of GPCRs in inflammation and other conditions.In this study we examined the potential for PAR Thus, in this study we examined the potential for PAR2 to synergize with TNFα in the activation of pro-inflammatory signalling responses such as JNK and p38 MAP kinase in a cell line (clone G) expressing both PAR2 and TNFR1 receptors. However to our surprise we found that activation of PAR2 was associated with a marked inhibition in TNFα stimulated JNK phosphorylation and to a lesser extent NFκB which was mediated by activation of the Gq/11 pathway via the activation of PKC and had a site of inhibition at the coupling of TNFR1 to receptor associated proteins. This extends a previous study from our laboratory examining purinergic P2Y receptors and suggests the existence of a novel paradigm for GPCR signalling, the inhibition of TNFα-stimulated JNK.Previous studies have demonstrated that activation of PAR22.12 activating peptide 2f-LIGKV-OH and the novel PAR2 peptide mimetic K-14585 were kind gifts from the Kowa Company Ltd. . The YM-254890 compound was a kind gift of Astellas Pharma. Inc, Japan.All materials used were of the highest commercial grade available and were purchased from Sigma  or Calbiochem , unless otherwise stated. The PKC inhibitor, GF109203X, was obtained from Calbiochem . Antibodies raised against phosphorylated forms of JNK and p38 MAP kinase were purchased from Biosource . Anti-IκBα, anti-JNK and p38 MAP kinase antibodies were obtained from Santa Cruz Biotechnology . Meanwhile, tumour necrosis factor-α (TNFα) was purchased from Insight Biotechnology . The PAR2.22 (clone G) were maintained in complete M199 medium containing 400 μg/ml of Geneticin for selection pressure and passaged using Versene (2% EDTA/PBS). EAhy-926 cells were produced by hybridizing human umbilical vein endothelial cells (HUVECs) with the epithelial cell line A549 Human skin epithelial cells NCTC2544 were maintained in M199 medium with Earl's salt supplement, 10% (v/v) foetal calf serum, L-glutamine (27 µg/ml), penicillin (250 U/ml), and streptomycin (25 mg/ml). NCTC2544 cells stably expressing human PAR2 at 37 °C.HUVECs were grown in endothelial basal media, supplemented with endothelial growth media (EGM-2) containing single aliquots  purchased from Cambrex. All experiments were performed between passages 3 and 7. All cells were kept in a humidified atmosphere containing 5% CO2.3Proteins were separated by 10% SDS–PAGE and transferred onto nitrocellulose. The membranes were blocked for non-specific binding for 2 h in 2% BSA (w/v) diluted in NATT buffer  Tween-20). The blots were then incubated overnight with 50 ng/ml primary antibody diluted in 0.2% BSA (w/v) in NATT buffer. The blots were washed with NATT buffer for 90 min and incubated with HRP-conjugated secondary antibody (20 ng/ml in 0.2% BSA (w/v) diluted in NATT buffer) for 2 h. After a further 90 min wash, the blots were subjected to ECL reagent and exposed to Kodak X-ray film.2.4For immuno-precipitation of TNFR1, equal amounts of pre-cleared cell lysates were incubated in Tris–HCl buffer, pH 7.4, containing 50 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% (w/v) Triton X-100, and 0.5% (w/v) Nonidet P-40, with 2 μg TNFR1 antibody, pre-coupled to protein G, for 3 h. Lysates were recovered by sequential washes in solubilisation buffer and in the same buffer lacking detergents. Precipitates were assessed for TRADD or RIP content using Western blotting.2.53VO4, 0.1 mM PMSF, 10 mg/ml aprotinin, 10 mg/ml leupeptin, and 1% (w/v) Triton X-100. Lysates were clarified by centrifugation for 5 min at 13,000 rpm and equal amounts of protein were incubated with 20 μg of GST-c-Jun-(5–89) immobilized on glutathione-Sepharose at 4 °C for 3 h. Beads were then washed three times in solubilisation buffer and twice in 25 mM HEPES buffer, pH 7.6, containing 20 mM β-glycerophosphate, 0.1 mM NaV3O4, and 2 mM dithiothreitol. Precipitates were then incubated with the same buffer containing 25 μM/0.5 μCi of ATP/[γ-32P] ATP in a final volume of 30 µl at 30 °C for 30 min. The reactions were terminated by the addition of 4× SDS-sample buffer and aliquots of each sample subjected to electrophoresis on 11% SDS-polyacrylamide gel electrophoresis. Phosphorylation of GST-c-Jun was then determined by autoradiography.To measure JNK activity, cells were stimulated as appropriate and the reaction terminated by rapid aspiration and the addition of ice-cold PBS. The cells were solubilised in 20 mM HEPES buffer, pH 7.7, containing 50 mM NaCl, 0.1 mM EDTA, 0.1 mM Na2.6P < 0.05, **P < 0.01).Where experimental data is shown as a blot, this represents one of at least 3 experiments and data represents the mean ± s.e.m. Statistical analysis was by one-way ANOVA with Dunnett's post test .Initially we sought to investigate the potential for synergy between PARInhibition of TNFα signalling was pathway specific , as no e2 was indeed required for tryspin and peptide mediated inhibition of TNFα-mediated JNK signalling (2 antagonist K-14585 was able to reverse the inhibition of JNK mediated by the human PAR2 activating peptide SLIGKV-OH (Panel B). Another candidate PAR, PAR4 expressed in the same cell type was without effect confirming the receptor specificity of the response (Panel C). Panel D illustrates that this inhibition is a feature of endogenously expressed PAR2, as pre-treatment of HUVECs with peptide reduced TNFα-stimulated JNK phosphorylation. Interestingly, P2Y2 stimulation mediated greater inhibition of TNFα stimulated JNK signalling, suggesting that other GPCRs exhibit the same phenomenon.We next sought to confirm that PARgnalling . Using e2 agonist 50 values of 6 μM (6.24 ± 1.319 μM) for SLIGKV-OH and approximately 2 μM (1.582 ± 0.3422 μM) for 2f-LIGKV-OH , similar to its ability to inhibit JNK stimulation in response to TNFα. In contrast, SLIGKV-OH was a less potent activator of JNK with an EC50 value of approximately 10 μM (10.12 ± 3.305 μM) in comparison to its ability to inhibit JNK (Panel C). Moreover, the difference in efficacy was marked, and SLIGKV-OH behaved as a full agonist in inhibiting TNFα signalling (% inhibition = 93.567 ± 6.882%) but was a partial agonist in stimulating JNK activity alone (% stimulation = 43.950 ± 6.482%).In addition, we further analysed the concentration dependency of not only SLIGKV-OH but also the substituted peptide 2f-LIGKV-OH originally identified by ourselves as a more potent PARLIGKV-OH , Panel A2. PAR2 is coupled to a number of signalling pathways via G-protein dependent and independent mechanisms. Initially, we found that we could mimic the inhibitory effect of PAR2 activation with PMA and that this effect could be reversed by micromolar concentrations of the PKC inhibitor, GF109203X, thereby implicating the involvement of PKC isoforms  of this inhibitor reversed the inhibitory effect of trypsin and SLIGKV-OH on TNFα-stimulated JNK activation (Panel B and C) and phosphorylation (Panel D). In contrast, another inhibitor Go6976 which preferentially inhibits the classical group of PKC isoforms was without effect (not shown) suggesting a potential role for the novel PKC isoforms.We then examined the potential signalling pathways which could be mediating the inhibitory effects of PARisoforms , Panel Aq/11 in the PAR2 mediated inhibitory effect .We next examined the potential site of inhibition within the TNFα-mediated cascade. We assessed the potential for PARreatment , Panel A42 inhibition of TNFα-induced JNK signalling. Our evidence strongly points to a Gq/11 and PKC-dependent cascade which functions to disrupt TNFR1 dependent signalling. Significantly, this inhibitory phenomenon is a feature of PAR2 in cells endogenously expressing the receptor and in response to activation of other GPCRs, namely P2Y2 receptors. Thus, it may have implications as to the regulatory role of GPCRs in a number of physiological and pathophysiological conditions.This study identifies a novel paradigm for PAR2 activation and other cytokines known to participate in inflammation such as TNFα, IL-1 and LPS through activation of selective TLRs. Indeed a recent study has demonstrated synergy between PAR2 and TLR-4 through the activation of Myd88 2 exhibits many features similar to cytokines regarding the activation of inflammatory mediator release, this study sought to characterise the differences and similarities of the signalling pathways mediated by both PAR2 and TNFα, and to examine the possibility of synergy between these two receptors. To our surprise we found that PAR2 activation was able to strongly mediate an inhibition of TNFα mediated JNK signalling in response to cellular activation by trypsin suggesting a PAR2 mediated response. Indeed the inhibitory effect of trypsin was mimicked with the peptides SLIGKV-OH and 2f-LIGKV-OH, but reversed by a novel PAR2 antagonist K14858, which we have recently characterised Initially, we sought to assess the potential of synergy between PAR2 and JNK. We have previously demonstrated that trypsin mediated activation of PAR2 results in an increase in JNK activity 2Our pharmacological studies also revealed the potential for agonist-mediated signalling in relation to PAR2 antagonist K-14585 2 mediated inhibition of TNFα signalling, we found that at higher concentrations, K14585 alone is able to activate both JNK (not shown) and p38 MAP kinase q/11 and one independent, mediating inhibition and activation of JNK respectively. Studies assessing JNK activation in response to GPCRs are limited; however, it has been shown that free βγ dimers and Gα12/13 subunits can activate JNK in a Rac1-cdc42 dependent manner 12 can also activate JNK via ASK1, independently of the activation of Rac1 and cdc42 Whilst this present study focuses on two agonist effects it is the first study to raise this potential when examining JNK activation. This model was also supported when examining the effect of the novel PAR2 mediated inhibition of JNK might occur. Our initial studies indicated that DAG-dependent PKC isoforms are involved in the response since PMA was able to mimic the effect of PAR2 activation, abolishing both JNK activity and phosphorylation. We found good reversal of PAR2 mediated inhibition using GF109203X, an inhibitor known to block both Ca2+-dependent and some novel PKC isoforms 2 cells were found to express the PKC isoforms, PKCα, β11, δ, ε and θ. This is typical of a number of keratinocyte cell types including NHEK and HaCat 2-mediated events including sensitization of the TRPV1 in rats and mice, and chloride transport in intestinal epithelial cells 2-dependent signalling events.We then sought to investigate the possible mechanisms by which PARq/11 in PAR2 mediated inhibition of JNK signalling. We used the novel Gq/11 inhibitory compound YM254890 which we have previously shown to be a very effective inhibitor of trypsin and peptide mediated [3H]IP accumulation 2 mediated JNK inhibition. Thus, DAG derived from Gq/11 activation is likely to drive PKC activation and subsequent inhibition of TNFα stimulated JNK activity. Previously, we have shown that Gq/11 plays a positive role in the regulation of PAR2 mediated NFκB phosphorylation and in DNA reporter activity 2 coupling to NFκB. Thus, these findings conflict with this present study showing the potential of Gq/11 to play a role in PAR2 mediated inhibition of TNFα stimulated IκBα loss. Interestingly, whilst we were able to demonstrate PAR2 mediated inhibition of TNFα induced IκBα loss this effect was not manifest further downstream, and pre-treatment of cells with peptide did not inhibit subsequent TNFα-stimulated NFκB reporter activity. Thus, because of the strong interactions of PAR2 with NFκB 2/TNFR1 is a loss in JNK activation only. We are currently assessing if receptors more poorly coupled to NFκB, for example P2Y2, can mediate an overall inhibition of NFκB activation.Our studies also revealed an important role for G2/Gq/11/PKC axis is essential in driving such disruption in response to PAR2. At this point the exact mechanism mediating disruption is unclear, no studies to date have identified any PKC phosphorylation sites within the TNFR1 complex, and thus it is possible that additional intermediate proteins may be involved.We also demonstrate for the first time receptor mediated driven disruption of TNFR1 binding to a number of associated proteins. Upon activation TNFR1 associates with a number of proteins including TRADD, RIP, TRAF2, MADD and FADD 2 would be able to reduce the damaging effects of TNFα during inflammation. In other cell types where PAR2 is expressed inhibition of TNFR1 signalling may be of significance in relation to cancer.The potential for GPCRs to mediate the inhibition of TNFα-mediated JNK signalling has clear physiological significance. For example, in the endothelium, activation of PAR52, a GPCR can disrupt TNFα mediated activation of the JNK pathway. This will provide valuable information regarding the role of GPCRs in the context of inflammation and other disease states.In conclusion we have demonstrated for the first time a novel mechanism whereby PAR1.2 mediated inhibition of TNFα activated JNK is via coupling to the heterotrimeric G-protein, Gq/11.PAR2.This inhibitory response requires members of the PKC family, and inhibits TNF activation of JNK by disrupting coupling of accessory proteins, such as RIP and TRADD to TNFR1.3.q/11 linked GPCRs, in particular by the P2Y2 receptor.This inhibitory effect is mediated by other GαSpecific points:


Previously, we reported that the “antioxidant” compound “mitoQ”  actually increased superoxide production by bovine aortic endothelial (BAE) cell mitochondria incubated with complex I but not complex II substrates.To further define the site of action of the targeted coenzyme Q compound, we extended these studies to include different substrate and inhibitor conditions. In addition, we assessed the effects of mitoquinone on mitochondrial respiration, measured respiration and mitochondrial membrane potential in intact cells, and tested the intriguing hypothesis that mitoquinone might impart fuel selectivity in intact BAE cells. In mitochondria respiring on differing concentrations of complex I substrates, mitoquinone and rotenone had interactive effects on ROS consistent with redox cycling at multiple sites within complex I. Mitoquinone increased respiration in isolated mitochondria respiring on complex I but not complex II substrates. Mitoquinone also increased oxygen consumption by intact BAE cells. Moreover, when added to intact cells at 50 to 1000 nM, mitoquinone increased glucose oxidation and reduced fat oxidation, at doses that did not alter membrane potential or induce cell toxicity. Although high dose mitoquinone reduced mitochondrial membrane potential, the positively charged mitochondrial-targeted cation, decyltriphenylphosphonium (mitoquinone without the coenzyme Q moiety), decreased membrane potential more than mitoquinone, but did not alter fuel selectivity. Therefore, non-specific effects of the positive charge were not responsible and the quinone moiety is required for altered nutrient selectivity.In summary, the interactive effects of mitoquinone and rotenone are consistent with redox cycling at more than one site within complex I. In addition, mitoquinone has substrate dependent effects on mitochondrial respiration, increases repiration by intact cells, and alters fuel selectivity favoring glucose over fatty acid oxidation at the intact cell level. Mitochondria are likely the predominant source of reactive oxygen species (ROS) in many cell types Concern over the contribution of ROS to vascular and degenerative disease, has led to attempts at antioxidant therapy. Efforts are now underway to develop effective antioxidant compounds targeted to mitochondria In past studies of bovine aortic endothelial (BAE) cell mitochondria, we demonstrated that a mitochondrial targeted CoQ compound termed “mitoQ”  markedly increased or decreased reactive oxygen species (ROS) generation depending on substrate provided for fuel During respiration on complex I substrates, superoxide appears to result from redox cycling of endogenous CoQ10 or exogenous analogs at Q-binding sites within complex I 2. Glucose oxidation through glycolysis and pyruvate dehydrogenase to acetyl CoA generates NADH. In contrast, fatty acid oxidation to acetyl-CoA generates FADH2 through the process of β-oxidation. Acetyl CoA from both sources feeds the TCA cycle. So, although mitochondrial fatty acid β-oxidation is more efficient in terms of total reducing equivalents generated per molecule, glucose oxidation compared to fat oxidation generates proportionally more NADH . So, we reasoned that if mitoquinone were more effective at generating oxygen radicals on substrates that produce NADH (complex I substrates) than those which generate FADH2, then mitoquinone might also increase respiration on complex I substrates more than complex II substrates. If so, mitoquinone might favor metabolism of nutrients that generate proportionately more NADH than FADH2 which might then translate to proportionately more (in the presence of mitoquinone versus absence) use of glucose than fat.Further, and perhaps of greater importance, we addressed the issues of whether mitoquinone might induce a substrate specific increase in mitochondrial oxygen consumption (as might be expected given the substrate specific effects on ROS) and whether this might be associated with alterations in cellular respiration and nutrient selectivity. This could follow based on the following reasoning. Complex I substrates generate NADH which donates electrons to complex I. In contrast, electron donation at other entry sites including complex II and the ETF-ubiquinone reductase requires FADHThe issue of fuel selectivity is of high interest in the fields of diabetes, obesity, and associated cardiovascular disease. In fact lack of metabolic “flexibility” such as the capacity to switch between fat and glucose oxidation has been implicated in cardiac complications of diabetes Here we report novel information covering two important issues. First we provide new data on the interactions of mitoquinone and rotenone at different concentrations of complex I substrates and discuss the implications for redox cycling at complex I. Second, we provide novel data demonstrating that mitoquinone does in fact alter respiration and fuel selectivity by intact cells. Finally we consider the effects of mitoquinone on mitochondrial membrane potential and whether such effects might affect fuel selectivity and/or cell toxicity.Mitoquinone and mitoquinol were synthesized from commercially available 11-bromoundecanoic acid and 2,3-dimethoxy-5-methyl-1,4-benzoquinone as described MitoQ consists of the quinone/quinol moiety of endogenous coenzyme Q, linked to a 10 carbon side chain, covalently bound to triphenylphosphonium. Control compounds included endogenous coenzyme Q, decylTPP consisting of triphenylphosphonium and the 10 carbon chain of mitoQ but without the quinone moiety, and decylQ consisting of the carbon chain and quinone/quinol moiety but without the targeting cation. The molecular structures of these compounds are included in supplementary material .Primary antibodies included mouse monoclonal anti-porin , goat polyclonals anti-actin and anti-histone deacetylase-1 (HDAC1), and rabbit anti-catalase IgG . Secondary antibodies consisted of goat anti-mouse, donkey anti-goat, and goat anti-rabbit (Santa Cruz).2 flasks and used between passages 6 and 12.BAE cells were grown in medium M199 (Invitrogen) supplemented with minimal essential medium amino acids (Invitrogen), penicillin/streptomycin (Invitrogen), minimal essential medium vitamins (Sigma), and 20% fetal bovine serum  as described Cells were washed with phosphate buffered saline (PBS) and scraped. Collected cells were homogenized using a Dounce homogenizer in ice-cold homogenization buffer  with 0.1% fatty acid-free bovine serum albumin (BSA). The homogenate was centrifuged at 1000×g for 10 min. The pellet was discarded and the supernatant was centrifuged again at 10,000×g for 10 min to obtain the mitochondrial pellet. The resulting pellet was then washed three times in homogenization buffer without BSA and resuspended in media as described below. Protein was determined by the Bradford method . Mitochondria prepared in this way were of good quality as documented by an increase in respiratory rate of 3.5–6 fold after addition of carbonyl cyanide p-[trifluoromethoxy]-phenyl-hydrazone (FCCP) as an uncoupler.Purity of mitochondria prepared in this way was documented by assessing mitochondrial and intact cell expression of the mitochondrial protein, porin; the cytoplasmic protein, catalase; the nuclear protein, HDAC1; and actin by immunoblot analysis. In our mitochondrial studies, all individual experiments (each n of 1) compare conditions within the same preparation, so the extent of purity would be the same for all conditions.Ten µg protein per lane were loaded and separated by 12.5% polyacrylamide gel electrophoresis and electroblotted to nitrocellulose membranes . For porin, blots were incubated with mouse anti-porin, 1∶25,000, in TTBS /2.5% BSA overnight at 4°C. For actin, blots were blocked with TTBS/5% milk at RT for 1 h and incubated in goat anti-actin, 1∶2000, in TTBS/2.5% BSA overnight at 4°C. For HDAC1, blots were incubated in goat anti-HDAC1, 1∶500, in TTBS/2.5% BSA overnight at 4°C. For catalase, blots were incubated in rabbit anti-catalase, 1∶2500 in TTBS/2.5% BSA overnight at 4°C. Following primary antibody incubations, blots were washed in TTBS, and incubated in secondary Ab, 1∶20,000, for 1 hour at room temperature in TTBS/5% milk. After secondary antibody incubations, blots were washed with TTBS and developed by enhanced chemiluminescence using a standard kit .2PO4, 2 mM MgCl2, 1 mM EGTA, 3 mM HEPES, pH 7.2 with 0.3% fatty acid free BSA). Isolated mitochondria (0.5 mg protein/ml) were incubated in the respiratory media and oxygen consumption quantified. To determine state 4 and state 3 respiration, oxygen consumption was continuously recorded with sequential additions of 5 mM succinate, 0.2 mM ADP, and finally 2.5 µM carbonyl cyanide p-[trifluoromethoxy]-phenyl-hydrazone (FCCP) to induce maximal chemical uncoupling using standard methodology as we have employed in the past Mitochondrial respiration was measured as we previously described 2O2 production was assessed as we previously described 2O2 to generate the fluorescent compound resorufin. ROS detected in this way derives largely from superoxide converted to H2O2 by matrix superoxide dismutase and released externally. As we previously showed by inhibitor analysis and by comparison to complex III superoxide detected by electron paramagnetic resonance spectroscopy (EPR) 2O2 detected in this way largely derives from superoxide released by complex I.Mitochondria were studied during state 4 respiration, under which circumstance oxygen radical formation is enhanced as electron flow leads to high potential unmitigated by ATP generation 2O2 standard curve ranging from 0–12 µM was prepared and included on each plate. Addition of substrates, mitoquinone, or diphenyleneiodinium chloride (DPI) to respiratory buffer did not affect the H2O2 standard curve. Addition of rotenone at 5 µM (maximum dose used) slightly raised the height of the standard curve at all points with no change in slope so the data were corrected for this factor. The lower doses of rotenone utilized in this work had no effect on the standard curve.Samples were prepared in 96-well plates containing 0.06 ml per well of respiratory buffer. Fluorescence was measured as we previously described 2O2. Addition of the ETS inhibitors rotenone or DPI or tested quinone or control compounds to mitochondria in the absence of substrate altered fluorescence no more than 5%.Addition of catalase, 500 units/ml, reduced fluorescence to below the detectable limit indicating specificity for H2 content across the column×oxygen content at atmospheric pressure (nmol/ml)×flow rate (ml/min)]/bead volume (ml). The apparatus and a sample run are depicted in supplementary material . Although we did not quantify the number of cells per bead volume the same preparations were used for mitoquinone and control in each experiment so any variation with cell number would be identical for mitoquinone and control. An example run is shown in supplemental material  wherein 2 released by perchloric acid as we previously described 6 µCi/pmol for cold oleate at 200 µM  and 20.1 µCi/pmol for cold oleate at 10 µM . Final specific activity of glucose was 0.0463×103 µCi/nmol for cold glucose at 5.5 mM .Cells were washed and then preincubated for 20 min in culture medium with 5.5 mM glucose, and either 10 µM or 200 µM oleate with 1.5% fatty acid-free BSA, and 1 mM carnitine in 12-well plates  containing 0.6 ml total volume per well. MitoQ (1 µM) or control compounds at the same concentration were added at the beginning of the 20 min preincubation (time −20 min) before addition of label at time 0. Separate groups of experiments were performed in the presence of oleate, 200 µM, and oleate, 10 µM. In each group of experiments glucose and oleate oxidation were assessed in parallel studies under the same conditions except for the addition of either [1–14C]oleic acid or D-[14C(U)]glucose. Cells were incubated for 120 min before trapping of CO6 µCi/pmol . Final specific activity of glucose was 0.0463×103 µCi/nmol .An additional group of experiments were carried out to determine the dose-response characteristics of the mitoquinone on fuel selectivity. These experiments were preformed in the same fashion except that cells were exposed to various concentrations of mitoquinone or vehicle in culture medium overnight (18 h) before addition of [1–14C]oleic acid or D-[14C(U)]glucose and 50 µM cold oleate at time zero (before the 120 minute incubation with label). Final specific activity of oleate was 5.61×10Cells were exposed to various doses of mitoquinone, decylTPP, CoQ10, or vehicle in culture medium overnight for 18 h. The potential sensitive probe, JC-1 was then added at a concentration of 0.5 µg/ml for one hour and washed with Earle's balanced salt solution before determining red and green fluorescence at excitation and emission wavelengths 544/590 and 485/520 respectively. The red to green ratio was calculated as an index of mitochondrial potential.Toxicity was assayed by two methods. First, the ability of cells to convert the redox dye resazurin into resorufin, a property lost by cells as metabolic activity is impaired Data were analyzed by ANOVA with post-tests as indicated.BAE mitochondria were highly pure as shown by the relative expression of the mitochondrial specific protein, porin; the cytoplasmic protein, catalase; the nuclear protein, HDAC1; and actin in mitochondrial and whole cell fractions . It is p2O2 production in the presence of the complex I substrates, glutamate and malate, while reducing H2O2 production in the presence of the complex II fuel, succinate. Here we show similar findings  indicating that the effect of mitoquinone is highly dependent on the rotenone concentration.In pilot experiments, we noted that rotenone inhibits the effect of mitoquinone on ROS in the presence of complex I substrates. However the effect seemed to change as different doses of these compounds were tested. In other experiments, we examined the effect of 5 µM DPI, which inhibits the FMN site of complex I, on ROS production as affected by mitoquinone at different rotenone concentrations. As shown, DPI reduced the effect of mitoquinone at all doses of rotenone. Since, there is evidence that DPI may also act on mitochondrial chloride channels Given the effects of mitoquinone on respiration in isolated mitochondria we extended these studies to whole cell respiration. This was done by perfusing cells grown on glass beads and measuring the difference in oxygen tension as recorded by electrodes placed proximal and distal to the perfused cells in a manner in which the two electrodes could be precisely calibrated to each other  and a second group in cells at 5.5 mM glucose and 10 µM oleate (before added label). Each individual data point actually represents the mean of triplicate determinations in cells grown in separate wells. The absolute values for glucose and oleate oxidation in the presence of vehicle were 4.96±0.85 nmol/well and 216±47 pmol/well in panels A and B, respectively, and 6.45±0.63 nmol/well and 11.8±4.3 pmol/well in panels C and D, respectively.As indicated, mitoquinone enhanced glucose oxidation  and inhiWe also determined the dose-response characteristics for the effect of mitoquinone on fuel selectivity. These studies were carried out as above except that the oleate concentration was 50 µM and the exposure time to mitoquinone or vehicle was overnight or 18 h. We found a significant dose response relationship extending well down into the nM range . The absCells were exposed overnight to 100 nM to 1.0 µM concentrations of mitoquinone, CoQ10, or decylTPP or to vehicle (ethanol) under the same conditions used to study fat and glucose oxidation . As showThese assays were done in order to test the possibility that mitoquinone or control compounds could induce cytotoxicity, which would raise concern regarding possible non-specific effects on mitochondrial or intact cell fuel utilization or on fuel selectivity. Cells were exposed overnight to 100 nM to 1.0 µM concentrations of mitoquinone, CoQ10, or decylTPP or to vehicle (ethanol), again under the same conditions used to study fat and glucose oxidation . We obseOur past work per se and is not specific for any particular NADH generating reaction. As shown in The major reason for the prooxidant effect of mitoQ is probably redox cycling within complex I. This was suggested by Doughan and Dikalov As shown in our current  and pastDPI acts proximal to the rotenone site in complex I blocking electron transfer at the FMN site. So the inhibition of the mitoquinone effect at all doses of rotenone by DPI  is compaIn contrast to its prooxidant effect in the presence of complex I substrates, mitoquinone decreased ROS production by mitochondria respiring on succinate . Under tOur current findings using JC-1 to assess potential as affected by mitoQ in intact cells differs from our past results which utilized the distribution of radiolabeled tetraphenylphosphonium inside and outside isolated mitochondria to assess potential 2O2 generated per mg per min was in the picomolar range ∶ubiquinone oxidoreductase. The later possibility is intriguing since inhibition might impair electron donation by FADHoquinone , it appeFor several reasons our work suggests that the effects of mitoquinone on ROS production, and respiration occur at complex I. These include the effect of mitoquinone to increase ROS and respiration selectively on complex I substrates  and 3, tWe examined fuel selectivity initially at high and low oleate concentrations, 200 µM and 10 µM, respectively, and subsequently at an intermediate concentration of 50 µM for our dose-response studies. These concentrations were arbitrarily chosen. For perspective, physiologic nonesterified (free) fatty acid concentrations in humans can range widely up to nearly 1 mM depending on nutrient and hormonal status and even higher is some disease states We considered the possibility that the effects of mitoQ on glucose use by intact cells  and 8 coOur current work has implications towards the development of mitoQ or related compounds as possible therapeutic agents useful as antioxidants. Possibly this approach might have benefit beyond antioxidant properties and extend to improved ability to use glucose countering the above mentioned effects of diabetes and insulin resistance. It is also important to recognize that although we observed prooxidant rather than antioxidant effects of mitoquinone with complex I substrates, the semiquinone generated by redox cycling of either endogenous CoQ10 or mitoQ may have the beneficial effect of acting as a chain breaking antioxidant In summary, we provide new data demonstrating that: 1) The prooxidant effects of mitoQ extend to respiration on pyruvate. 2) The prooxidant effects are perturbed by rotenone, but in a complex fashion consistent with action at more than one Q binding site. 3) Importantly, mitoquinone increases mitochondrial respiration in substrate-specific fashion, increases intact BAE cell respiration, and imparts fuel selectivity in the intact cell favoring glucose while inhibiting fat oxidation. 4) MitoQ decreases membrane potential, which likely explains its effect to reduce ROS during reverse electron transport. However, the prooxidant effects of mitoQ and the effects on fuel selectivity are not explained by decreased membrane potential and require the quinone moiety of the compound.Methods S1Supplemental information(0.28 MB DOC)Click here for additional data file.