CDK2 and PKA were found to participate in p19INK4d phosphorylation process and that they would mediate serine 76 and threonine 141 modifications respectively. Nuclear translocation of p19INK4d induced by DNA damage was shown to be dependent on serine 76 phosphorylation. Most importantly, both phosphorylation sites were found to be crucial for p19INK4d function in DNA repair and cell survival. In contrast, serine 76 and threonine 141 were dispensable for CDK4/6 inhibition highlighting the independence of p19INK4d functions, in agreement with our previous findings. These results constitute the first description of the activation mechanism of p19INK4d in response to genotoxic stress and demonstrate the functional relevance of this activation following DNA damage.DNA damage triggers a phosphorylation-based signaling cascade known as the DNA damage response. p19INK4d, a member of the INK4 family of CDK4/6 inhibitors, has been reported to participate in the DNA damage response promoting DNA repair and cell survival. Here, we provide mechanistic insight into the activation mechanism of p19INK4d linked to the response to DNA damage. Results showed that p19INK4d becomes phosphorylated following UV radiation, β-amyloid peptide and cisplatin treatments. ATM-Chk2/ATR-Chk1 signaling pathways were found to be differentially involved in p19INK4d phosphorylation depending on the type of DNA damage. Two sequential phosphorylation events at serine 76 and threonine 141 were identified using p19INK4d single-point mutants in metabolic labeling assays with  DDR consists of an intricate signaling network in which complex DNA surveillance programs play a key role The cell cycle progression is driven by the activity of cyclin-dependent kinases (CDKs) and is negatively regulated by INK4 and Cip/Kip inhibitory proteins Despite these findings, the regulation of p19 activity in the DDR remains unknown. We hypothesized that post-translational modifications on p19 could be taking part in regulating this specific function. Here, we report the activation mechanism of p19 in response to DNA damage. It is shown that this event is dependent on the ATM/ATR signaling pathways and occurs in a sequential manner at serine 76 (S76) and threonine 141 (T141). These two identified phosphorylation sites would be direct substrates for CDK2 and PKA kinases respectively. Moreover, DNA damage induced p19 nuclear translocation requires S76 phosphorylation. And finally, both phosphorylation sites are shown to be crucial for p19 function in DNA repair and cell survival but dispensable for CDK4/6 inhibition. These results position p19 in a novel context as a downstream target of the DDR signaling pathways, provide mechanistic insight into the activation mechanism of p19INK4d in response to DNA damage and demonstrate the functional relevance of this activation following genotoxic stress.6 cells were seeded in 60-mm plates and transfected with 4 µg of wild type or mutant p19 expression plasmids. 24 h after transfection, cells were treated as indicated in each experiment. For UDS, [3H]thymidine incorporation and caspase-3 activity assays, WI-38 cells were plated in 35-mm dishes at a density of 1×106 cells/well in 2.5 ml of medium. After a 24 h attachment period, each well was transfected with a mixture containing 2 µg of wild type or mutant p19 expression plasmids and 0.5 µg of pBabePuro per plate. Twenty-four hours after transfection, 2.5 µg/ml puromycin were added for 48 h to select for transfected cells.WI-38 cell line was grown in minimum essential medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin, 100 mM non-essential aminoacids, and 2 mM glutamine at 37°C in a humidified 5% CO2 atmosphere. HEK-293 cells were grown in Dulbecco's modified Eagle medium supplemented as described above. Transfections were performed using Lipofectamine 2000 Reagent (Invitrogen). Approximately, 2×10Antibodies: p19 , p19 , V5 , V5 . CDK2 , PKAc, histone H3 . GAPDH . Anti-mouse and anti-rabbit secondary antibodies conjugated with horseradish peroxidase were purchased from SIGMA .2), 254 nm (range 240–280 nm) at room temperature using a Philips ultraviolet lamp (TUV15WG15T8) calibrated to deliver 0.25 mJ/cm2 s. Following UV irradiation, medium was replaced and cells were further incubated for the indicated times. For each experiment, control cells were treated identically except for UV light exposure.Cells were irradiated in open dishes with UV  were incubated with the appropiate antibody for 2 h, followed by O.N. incubation with protein AG agarose beads at 4°C. After washing three times with RIPA buffer, samples were analyzed by immunoblotting or SDS-PAGE. Dried gels were exposed to a radiographic intensifying screen by Fujifilm and scanned directly using a Bio-Imaging Analyzer Fujifilm BAS-1800II.WI-38 cells were grown in 60-mm dishes and treated as indicated. Before treatment, cells were incubated with 0,5 mCi sodium orthophosphate-Construction of p19 mutants is described in Supporting Information .in vivo phosphorylation as described previously. ASCDK1: 5′ tattttggtattatcttccatagttag 3′; ASCDK2: 5′ccaacttgaaacaatcttgccgcctccc 3′.Antisense oligonucleotides (AS) complementary to either human CDK1 or CDK2 (corresponding to bases +129 to +155 and +46 to +73 respectively) were transfected with Lipofectamine 2000. At a final concentration of the AS was 1 µM. After 12 h, the medium was replaced by fresh medium containing 1 µM of the corresponding AS. Cells were treated for http://www.ks.uiuc.edu/Research/vmd/). Simulations of p19 phosphorylations were obtained using AMBER software. XY graphs were done using XMGRACE utility. .Analysis of p19 structure was performed using the VMD programmed . NetphosK 1.0 server was used for kinase-specific prediction of phosphorylation sites http://www.cbs.dtu.dk/services/NetPhosK/).Netphos 2.0 server Protein sequences were aligned using T-Coffee multiple sequence alignment tool 32P-labeled probes to CDK1, CDK2 and β-ubulin. To detect CDK1 mRNA, a 28-mer ODN was synthesized complementary to bases +124 to +151 of human p19 mRNA. To detect CDK2 mRNA, a 29-mer ODN was synthesized complementary to bases +45 to +73 of human p19 mRNA. To detect β-tubulin a 22-mer ODN was synthesized complementary to bases +174 to +195 of human tubulin beta 3 mRNA. ODN were 5-end-labeled using [γ-32P] ATP and T4 polynucleotide kinase. Hybridization was carried as previously described Total cellular RNA was isolated from cultures as described previously 32P]ATP  per 10 µl of 2× reaction buffer). Samples were incubated for 20 min at 37°C. The reaction was stopped by addition of 7.5 µl of 5× sample buffer and 5 min incubation at 95°C. Histone H1 from calf thymus  was used for kinase activity control. Samples were electrophoresed on 12% denaturing gels. Gels were dried on Whatman paper, exposed to a radiographic intensifying screen by Fujifilm and scanned directly using a Bio-Imaging Analyzer Fujifilm BAS-1800II. In a similar assay, phosphorylation of a p19 derived peptide containing the surrounding sequence of S76  was tested. As control for CDK2 activity, an histone H1 derived peptide . Following the reaction, samples were processed according to the phosphocellulose paper method. As a negative control, the reaction was conducted without substrate.CDK2 kinase assay was performed as described by Giono et. al. in vitro phosphorylation assays were performed in a final volume of 40 µl of reaction buffer  0,1 mM ATP, 100 µg/ml PMSF, 60 µg/ml aprotinin, 1 mM sodium orthovanadate) plus 5 µg of GST-p19 with or without H-89 (100 µM). After 15 min at 30°C, samples were electrophoresed on 12% denaturing gels. Gels were dried on Whatman paper, exposed to a radiographic intensifying screen by Fujifilm and scanned directly using a Bio-Imaging Analyzer Fujifilm BAS-1800II. In a similar assay, phosphorylation of a p19 derived peptide containing the surrounding sequence of threonine 141  was tested. As control for PKA activity, Kemptide was used as substrate . Following the reaction, samples were processed according to the phosphocellulose paper method. As a negative control, the reaction was conducted without substrate.The Co-immunoprecipitation assays were performed by transfection of pcDNA4cp19wt in WI-38 cells. A total of 500 µg of proteins were immunoprecipitated with 3 µl of anti-V5 antibody and 30 µl of 50% slurry of protein A/G agarose beads (SIGMA). The beads were washed three times with RIPA, resuspended in 30 µl of 2× sample buffer, and heated to 95°C for 5 min. Proteins were resolved in 15% polyacrylamide gels and analyzed by immunoblotting.2) or 10 µM β-amyloid peptide for 12 h. Cells were then harvested with lysis buffer  incubated for 30 min at 37°C and centrifuged at 12,000× g for 20 min. The activity of caspase-3 in 150 µl of cell lysate was determined using 100 µM of the synthetic caspase-3 substrate Ac-DEVD-pNA (Sigma) in reaction buffer  in a final volume of 300 µl and incubated at 37°C during 4 h. Color development was measured at 405 nm. Caspase activity was estimated as A405/µg protein.Cells were treated with UV  (Amersham Biosciences) for 6 h. Cells were washed three times with cold PBS, harvested, and centrifuged at 3000×g for 5 min. The cellular pellet was lysed with 5% trichloroacetic acid (TCA) for 30 min, centrifuged and washed twice with cold H2O2. The pellet was resuspended in 150 µl 1 M NaOH for 1 h at room temperature. Incorporated radioactivity was quantified by scintillation counting and DNA synthesis expressed as dpm/µg protein.Twenty four hours after transfection, cells were incubated with 1 µCi/ml thymidine. At the indicated times, cells were washed three times with cold PBS, harvested and collected at 3000×g for 5 min. Cells were lysed with 5% TCA for 30 min. and centrifuged at 10,000×g for 10 min. Pellet was washed twice with cold PBS and resuspended in 1 M NaOH. The incorporated radioactivity was quantified by scintillation counting. Unscheduled DNA synthesis was expressed as dpm/µg protein.Twenty four hours after transfection, cells were washed with PBS and growth medium was replaced by arginine-free medium containing 1% FBS which was renewed after 24 h. Inhibition of DNA semiconservative synthesis was confirmed under these conditions. Cells were treated with UV  or at both sites, S76 and T141 (p19S76E/T141E). In accordance with the hypothesis of a sequential phosphorylation, the phosphomimetic mutation at S76 enabled the phosphorylation of p19S76E mutant at T141  direct comparison between p19 and p19p average structures showed significant differences . Up to 8Overall, we propose a sequential phosphorylation model for p19 in which modification at S76 would enable a second phosphorylation event at T141. The phosphorylation-induced structural changes could have functional implicancies for p19 in the DNA damage response.. Since we demonstrated that p19 phosphorylation is induced by all three DNA damaging agents, we hypothesized that the ATM/ATR pathway could be involved in this process. To examine this possibility, caffeine, a specific PIKKs inhibitor, was used in the analysis of p19 phosphorylation in vivo. Caffeine treatment effectively prevented p19 phosphorylation .In vivo p19 phosphorylation was not affected by ASCDK1 treatment. In contrast, a decrease in the phosphorylation was found following ASCDK2 treatment suggesting a role for CDK2 in this process.We next aimed to identify the CDK family member necessary for p19 phosphorylation acting in this process. Since CDK5 activity was only reported in neural cells, the involvement of CDK1 and CDK2 kinases was examined in the cell lines tested. Specific antisense oligonucleotides were used to down-regulate either CDK1 (ASCDK1) or CDK2 (ASCDK2). The efficiency and specificity of the antisense oligonucleotides was first tested by Northern blot  or T141 (p-T141: RDARGLTPLELA) phosphorylation sites were employed to test the direct activity of CDK2 or PKA correspondingly. Results showed that CDK2 was able to efficiently phosphorylate the p-S76 peptide  displayed similar abilities to block cell proliferation compared to p19wt . TherefoThe DDR involves complex signal transduction pathways that regulate DNA repair and cell death mechanisms to restore DNA integrity or to eliminate the damaged cell. We have previously reported that p19 participates in the DDR being necessary for an efficient repair of the DNA damage Two mechanisms are essential in response to genotoxic stress to maintain genome integrity: DNA repair and apoptosis. As part of the DDR, when the DNA damage is too severe to be repaired, cell death programs are activated to eliminate the cell irreversibly affected. It was previously reported that p19 overexpression significantly decreases the apoptosis induced by UV light, cisplatin and β-amyloid peptide Altogether, these findings are consistent with a regulation pathway in which S76 and T141 phosphorylation is critical for the reported function of p19 linked to the response to DNA damage.In response to DNA damage, conserved checkpoint mechanisms trigger multiple events that coordinate cell cycle progression, DNA repair and cell death in order to restore DNA integrity or to eliminate the irreversibly damaged cell. The present study uncovers the activation mechanism of p19 in response to DNA damage. The results show that p19 is a downstream target of the main DDR signaling pathways, ATM/Chk2 and ATR/Chk1. It was demonstrated that p19 function involved in DNA repair and cell survival is modulated by a sequential phosphorylation at serine 76 and threonine 141 dependent on CDK2 and PKA. A previous work reported p19 phosphorylation under basal conditions in U2OS cell line and suggested serines 66 and 76 as phosphorylation sites The structural and dynamic consequences caused by the phosphorylations on p19 were analyzed by Molecular Dynamics (MD). Simulation of S76 phosphorylation (p19p) showed a main increase in the mobility of the third ankyrin motif, where the negative charge was located, but also mobility differences in the fourth and fifth ankyrin repeat were observed. The experimental data indicated a sequential phosphorylation at S76 and T141 aminoacids. Supporting the sequential phosphorylation results, the structural changes in the fifth ankyrin repeat induced in p19p might be necessary to enable T141 phosphorylation. The main differences found when both, S76 and T141, phosphorylations were simulated are located in the fifth repeat. We propose that the additional changes in the structure promoted by the second phosphorylation could be involved in the interaction of p19 with proteins related to DDR mechanisms and then might be critical for p19 function. Further experimental studies are being conducted to identify p19 interactors and verify this possibility.In vitro assays confirmed direct CDK2-mediated phosphorylation of p19. Adding to this, CDK inhibition prevented DNA-damage-induced phosphorylation of endogenous p19. Specific down-regulation of CDK2 impaired p19 phosphorylation. Together, these results show the dependence of p19 phosphorylation on CDK2 function and strongly suggest the direct action of this kinase on this protein. In vivo CDK inhibition also blocked the phosphorylation of p19T141A and p19ANKless mutants. Since only two residues, S76 and T141, become phosphorylated after DNA injury these observations also indicate that S76 might be the specific target site for CDK2. T141 in p19 was shown to be embedded within a PKA consensus motif. PKA has been shown to exert an antiapoptotic effect in different cell lines. In addition, PKA activity was implicated in the activation of the processivity factor PCNA and in the nuclear translocation of DNA-PK, two critical proteins in DNA repair in vitro and in vivo supported the direct action of PKA on p19. Moreover, the decreased phosphorylation observed for endogenous p19 after H-89 treatment was consistent with a reduced phosphorylation of p19T141A and p19ANKless mutants. Even more, no further reduction in p19T141A or p19ANKless phosphorylation was found by PKA inhibition, suggesting that the action site of this kinase had already been eliminated in these mutants. Taken together, these findings support p19 phosphorylation by PKA in response to DNA damage and point out to T141 as the target site for this kinase.It is well established that inhibition of CDK activity, one of the main actions promoted by checkpoint responses, leads to cell cycle arrest and provides time for DNA repair. However, increasing evidence supports an active role for CDKs in the DDR. Overall CDK activity was reported to be necessary for an efficient DDR activation after γ-irradiation induced DNA damage The regulation of protein localization provides cells with a convenient way to modulate their functions. p19 does not contain the standard basic monopartite or bipartite nuclear localization signal usually found in nuclear proteins Protein phosphorylation is a widely used mechanism to selectively modulate protein activity. We then investigated if phosphorylation had a functional relevance on p19. The expression of p19 mutants lacking S76 and/or T141 promoted cell cycle arrest at similar levels to those observed for wild type p19. These results indicate that neither S76 nor T141 are necessary for p19 inhibition of CDK4/6 kinases. Previous works based on crystal structure analysis showed that binding to CDK6 involves mainly ankyrin domains I–III of p19. In accordance with our findings, threonine 141 is positioned within the fifth ankyrin repeat and then would not participate in the interaction with CDK. Moreover, S76, located in the third ankyrin repeat, was not described to be implicated in CDK binding by NMR studies. In summary, our results uncover the activation mechanism of p19 implicated in the response to DNA damage. We propose that the phosphorylation of specific sites might induce conformational changes in p19 necessary for the correct subcellular localization and for the interaction with DDR proteins. Mutations in DDR critical genes that lead to impaired genome stability, increased cancer susceptibility or enhanced cell death reflect the importance of a proper DDR. Consequently, a comprehensive knowledge of the DDR pathways becomes essential to understand disease development and might contribute to establish more efficient therapeutic approaches.Materials and Methods S1Description of the mutagenesis strategy used to construct p19 mutants.(DOC)Click here for additional data file.Figure S1p19 immunoprecipitation specificity. WI-38 fibroblasts were labeled with [32P]-orthophosphate and treated with β-amyloid peptide (20 µM), cisplatin (10 µM) or UV light (4 mJ/cm2) for 3 hours. Equal amounts of whole cell extracts were subjected to immunoprecipitation with anti-p19 antibody  or anti-V5 antibody as a control antibody . The immune complexes were analyzed by SDS-PAGE and autoradiography  or immunoblotting .(TIF)Click here for additional data file.Figure S2Prediction of p19 phosphorylation sites. p19 protein sequence was analyzed for the presence of potential phosphorylation sites using the bioinformatic tool Netphos 2.0 server. Tables show serine predictions (A) or threonine predictions (B), no putative tyrosine phoshorylation sites were found. (C) Graph shows the score of the predicted phosphorylation sites. Pos, position of the potential phosphorylation site.(TIF)Click here for additional data file.Figure S3Prediction of kinase specific phosphorylation sites in p19. p19 protein sequence was analyzed for the presence of kinase specific phosphorylation sites using the bioinformatic tool NetphosK 1.0 server with evolutionary stable sites filter (ESS filter). Table shows the position of the putative phosphorylation sites for the indicated kinases. .(TIF)Click here for additional data file.Figure S4p19 phosphorylation is not abolished by high concentrations of PKA inhibitor. WI-38 fibroblasts were incubated with the indicated concentrations of H-89 for 1 hour, and then treated with cisplatin (10 µM) for 2 hours and endogenous p19 phosphorylation analyzed by autoradiography.(TIF)Click here for additional data file.Figure S5S76 and T141 are not involved in the cell cycle function of p19. Proliferation status of cells overexpressing p19wt or p19 phosphorylation deficient mutants. WI-38 fibroblasts were transfected with p19wt or the indicated p19 mutants. Cells were incubated with [3H]-thymidine for 5 hours and the lysates were tested for tritium incorporation. Bars represent the mean ± s.e.m of three independent experiments performed in triplicate. Student's t-test was used to compare control sample (none) with p19wt or p19 mutant samples. .(TIF)Click here for additional data file.Figure S6Phosphorylation of S76 and T141 is required for p19 function in DNA repair. (A) DNA repair ability of cells overexpressing p19wt or p19 phosphorylation deficient mutants. WI-38 fibroblasts were transfected with p19wt or the indicated p19 mutants. Cells were maintained in an arginine-free medium containing 1% fetal bovine serum during 48 h. β-amyloid peptide (20 µM) was added to the medium and cells were incubated with [3H]-thymidine for 10 hours. Cell lysates were tested for Unscheduled DNA Synthesis assay (UDS). Bars represent the mean ± s.e.m of three independent experiments performed in triplicate. Student's t-test was used to compare β-amyloid peptide-treated control sample (none) with β-amyloid peptide-treated p19wt or p19 mutant samples. . Protein expression was analyzed by immunoblot. (B) Similarly as in (A) but overexpressing the phosphomimetic p19 mutants.(TIF)Click here for additional data file.Figure S7Phosphorylation of S76 and T141 is necessary for p19 function in apoptosis. β-amyloid peptide-dependent apoptotic response of cells overexpressing p19wt or the phosphorylation deficient mutants, p19S76A and p19T141A. WI-38 fibroblasts were transfected with p19wt or the indicated p19 mutants. β-amyloid peptide (20 µM) was added to the medium and following 12 hours cell lysates were tested for caspase-3 activity. Results are expressed as percentage of caspase-3 activity with respect to basal activity of cell lysates nontransfected and without β-amyloid peptide-treatment, which was set to 100. Bars represent the mean ± s.e.m of three independent experiments performed in triplicate. Students t-test was used to compare, β-amyloid peptide-treated control sample (none) with β-amyloid peptide-treated p19wt or p19 mutant samples (*p<0.005).(TIF)Click here for additional data file.Figure S8Conservation of p19 phosphorylation sites in different mammalian species. p19 protein sequences from the indicated mammals were align using T-Coffee multiple sequence alignment tool. Arrows indicate the position of S76 and T141 from p19 human sequence.(TIF)Click here for additional data file.Figure S9Alignment of protein sequences of the INK4 family members. Protein sequences were align using T-Coffee multiple sequence alignment tool. Arrows indicate the position of S76 and T141 from p19 protein sequence. .(TIF)Click here for additional data file.


Activation of NADPH oxidase is required for neutrophil extracellular trap (NET) formation. Protein kinase C (PKC) is an upstream mediator of NADPH oxidase activation and thus likely to have a role in NET formation.Pharmacological inhibitors were used to block PKC activity in neutrophils harvested from healthy donor blood.Pan PKC inhibition with Ro-31-8220 (p<0.001), conventional PKC inhibition with Go 6976 (p<0.001) and specific PKCβ inhibition with LY333531 (p<0.01) blocked NET formation in response to PMA. Inhibition of novel and atypical PKC had no effect. LY333531 blocked NET induction by the diacylglycerol analogue OAG  (p<0.001).Conventional PKCs have a prominent role in NET formation. Furthermore PKCβ is the major isoform implicated in NET formation. Neutrophil granulocytes are key cells of the innate immune system with a primary function of killing invading microorganisms such as bacteria, fungi and parasites to prevent pathogenic spread and invasion,2. Once Shigella flexneri, Staphylococcus aureus, Salmonella thyphimurium, Streptococcus pneumoniae and the fungus Candida albicans[NET formation is known to be stimulated by specific cytokines ), bacterial products ) and importantly by clinically relevant pathogens such as  albicans. Stimula albicans.The PKC isozyme family is comprised of conventional, novel and atypical isoforms. There aDihydrorhodamine (DHR), dimethyl sulfoxide (DMSO), diphenyliodonium (DPI), Phorbol 12-myristate 13-acetate (PMA), Ro-31-8220, PKCζ pseudosubstrate myristoyl trifluoroacetate (PKCζ inhibitor) and SYTOX green were purchased from Sigma-Aldrich ; Rottlerin, Gö 6976 and LY333531 were from Calbiochem (Merck) .Peripheral blood neutrophils were isolated from healthy human volunteers according to Lothian Research Ethics Committee approvals #08/S1103/38 via dextran sedimentation and Percoll™ discontinuous gradients as described. Informe4 cells/well) in HBSS containing Ca2+, Mg2+ and Hepes (20 mM) were aliquoted (180 μl) into 96 well plates and left to settle for 30 min at 37°C. The inhibitors Ro-31-8220, DPI, rottlerin, PKCζ inhibitor, Gö 6976 and PKCβ inhibitor were added at appropriate concentrations to wells in duplicates and incubated for 30 min before adding PMA. The final volume in each well was 200 μl. Plates were incubated for 4 h and then SYTOX green , a cell-impermeable nucleic acid stain, with an excitation/emission maxima of 504/523 nm to give a green fluorescent light, was added and NET formation was observed by measuring mean fluorescence in 96 well plates. In some experiments 1-oleoyl-2-acetyl-sn-glycerol (OAG) was used to stimulate cells in place of PMA. Results were evaluated by measuring the mean fluorescence in 96 well plates after the subtraction of background fluorescence. Cells were also visualised by fluorescent microscopy carried out on a Zeiss Axiovert S100 fluorescent microscope  and an Evos fl inverted microscope .Neutrophils (5×10Data were assessed by one way ANOVA followed by a post-hoc Dunnett’s test. The data were expressed as mean ± standard error of the mean (SEM), and values of p < 0.05 were considered statistically significant. All statistics were performed using GraphPad Prism 5 software .2=0.98), data not shown. Therefore total fluorescence was utilised as a reliable screening assay in further experiments to allow a range of inhibitors to be compared, before confirmation with gold-standard microscopic validation. This test was reproducible with an average inter-assay coefficient of variation of 14.3%. As almost maximal NET formation was gained with 10 nM PMA  for 30 min and then treated with 10 nM PMA. Both Ro-31-8220 and DPI, completely inhibited PMA induced NET formation , rottlerin (Novel), PKCζ psuedosubstrate   was employed Figure . LY33353To elucidate the downstream effects of PKCβ inhibition we assessed the effects of LY333531 on oxidative burst as measured by DHR fluorescence on flow cytometry. LY333531 reduced NADPH oxidative burst at the same concentrations required to reduce NET formation, with a partial but significant knock down of activity at 100 nM (p<0.01) and a complete knockdown at 1 μM (p<0.001) in comparison to the positive controls of DPI and Ro-31-8220  fluorescence as described previously. NeutropThe results clearly show that NET formation induced by PMA is PKC and NADPH oxidase dependent. NET formation was blocked by both Pan-PKC inhibition and conventional-PKC inhibition. Furthermore, a specific PKCβ inhibitor (LY333531) also blocked NET formation. LY333531 has high selectivity for PKCβ over other conventional isoforms (IC50 of around 5 nM) with a 60 fold selectivity for PKCβ over PKCα. At high2-) and nitric oxide (NO) are fundamental responses of the neutrophil to inflammatory stimuli and pathogens. NET formation is dependent on NADPH oxidase activation and consequently on the generation of 02- which can be blocked by DPI. DPI inhibits NADPH oxidase by binding to specific subunits in the enzyme complex and preventing electron flow and 02- production[2- which are then spontaneously converted to H2O2. Interestingly, p47phox has to be phosphorylated to acquire a conformational rearrangement to expose the domains that are important for the NADPH oxidase function, and this phosphorylation is mediated by PKC[Oxidative burst and the generation of reactive oxygen species including superoxide anions  may offer a novel anti-inflammatory strategy.The authors declare that they have no competing interests.RDG, CDL, AM, FL and KM carried out the experiments. RDG, CH, DJD and AGR designed the experiments and provided a critical review of methods. RDG drafted the manuscript. All authors read and approved the final manuscript.RDG is a Wellcome Trust Fellow (093767). DD is an MRC Senior Research Fellow (G1002046). This work was also funded by the Wellcome Trust  and the MRC .


Depot antipsychotic injections are an important tool for the management of patients with schizophrenia who have difficulty with adherence to oral medication. However, pain and discomfort at the injection site can be a potential impediment to the use of these long-acting formulations. We report here the results of a pooled analysis of injection site-related adverse events (AEs) collected during treatment with the olanzapine long-acting injection (olanzapine LAI).Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition (DSM-IV) or Fourth Edition, Text Revision (DSM-IV-TR) diagnosis of schizophrenia or schizoaffective disorder and were between the ages of 18 and 75. Doses ranged from 45 to 405 mg olanzapine LAI, and injection intervals were 2, 3, or 4 weeks. Events were evaluated for severity, timing, possible risk factors, and outcome. A criterion of p < .05 for statistical significance was used for all tests.Unsolicited injection site-related AEs were pooled from 7 olanzapine LAI clinical trials conducted in patients between March 2001 and December 2010. All patients had a A total of 1752 patients received at least 1 olanzapine LAI injection. Of these, 92 patients (5.3%) reported at least 1 injection site-related AE, with “pain” being the most common type (2.9%). Most events were mild (81.4%) and the median duration was 3 days. Four patients (0.2%) discontinued due to injection site-related AEs. Dose volume and body mass index did not appear to affect the probability of injection site-related AEs. However, patients who experienced a post-injection delirium/sedation syndrome event (n = 37) were more likely to have or have had an injection site-related AE at some time during the study. Incidence of injection site-related AEs appeared to decrease over time. In 94.2% of the injection site-related AEs, no specific treatment or concomitant medication was reported; in 9 cases, patients received pharmacologic treatment for reaction, mass, abscess, rash, or pain.Injection site-related AEs with olanzapine LAI were generally mild. The incidence and nature of these injection site-related AEs were generally similar to those occurring during treatment with other injectable antipsychotics.NCT00094640, NCT00088478, NCT00088491, NCT00088465, and NCT00320489.ClinicalTrials.gov ID; URL:  Despite advances in the treatment of schizophrenia with the use of newer atypical antipsychotic agents, treatment nonadherence continues to be a problematic and costly issue. As many as one-third of outpatients are reported to be completely noncompliant, and another third are reported to be only partially compliant . MoreoveLong-acting depot injections of antipsychotic agents are important in the treatment of schizophrenia because they may help mitigate treatment noncompliance and its consequences. However, pain and discomfort at the injection site can be a potential impediment to the use of these depot formulations. Injection site adverse events (AEs), including injection site pain, have been reported with conventional and atypical depot agents -6. HowevOlanzapine long-acting injection (LAI) is a depot antipsychotic formulation consisting of a pamoate salt of olanzapine that is administered by deep intramuscular injection every 2 to 4 weeks . The effThe purpose of this paper is to determine the extent and nature of injection site-related AEs in patients treated with olanzapine LAI. An analysis of pooled data from 7 olanzapine LAI studies will be presented.N = 134, Study F1D-EW-LOBS), a 2-month pharmacokinetic study , a receptor occupancy study   reporting an injection site-related AE) and thus were not comparable with those of the other studies, which relied solely upon spontaneous reporting of AEs.This pooled analysis is based on 7 olanzapine LAI clinical trials that were conducted in patients between March 2001 and December 2010: a single-dose pharmacokinetic study (MC-HGJZ) , a 24-weMC-HGKB) . In addiDiagnostic and Statistical Manual of Mental Disorders, Fourth Edition (DSM-IV) or Fourth Edition, Text Revision (DSM-IV-TR), were between the ages of 18 and 75, and had at least 1 olanzapine LAI injection [All patients had a diagnosis of schizophrenia or schizoaffective disorder, as defined by the njection . ExclusiPatients received their injections after completion of all efficacy and safety assessments at that visit. Injections were generally administered into alternating sides of the buttocks from visit to visit, and administrators were advised not to massage the injection area after injection. Injections were administered using a 19-gauge 1.5-inch (or 35-mm) needle; a 2-inch (or 50-mm) needle could be used for obese patients. Doses ranged from 45 to 405 mg olanzapine LAI, and injection intervals could be 2, 3, or 4 weeks, depending on the specific study. Injections given at 2-week intervals could not exceed a dose of 300 mg.p < .05 for statistical significance was used for all tests.All analyses were performed using SAS version 9.1 . Only injection site-related AEs were assessed . The Cochran-Armitage test for trend was used to assess whether there was any increasing or decreasing trend in the relationship between the presence of injection site-related AEs and injection volume , or body mass index . Because a patient’s BMI category could change over the course of a study, BMI at the first injection was used for patients who never reported an injection site-related AE, but for patients with injection site-related AEs BMI at the first occurrence was used. Chi-square tests were used to compare any 2 proportions. Fisher’s Exact test was used to compare the percentage of patients experiencing both PDSS and at least one injection site-related AEs with the percentage of patients who experienced PDSS only. A criterion of From the 7 pooled studies, a total of 1752 patients received at least 1 olanzapine LAI injection . Of these 1752 patients, 92 (5.3%) reported at least 1 injection site-related AE  were severe. Four patients (0.2%) discontinued due to injection site-related AEs . None of these subjects had experienced a previous event.p = .149).There was no significant relationship between dose volume and the probability of an injection site-related AE (p = .041).An analysis of injection site-related AEs and PDSS events demonstrated that the percentage of patients who experienced a PDSS event was greater among those who, at some point during the study, also experienced an injection site-related AE versus those who did not .We also tested if the percentage of patients experiencing an injection site-related AE was associated with BMI. The percentages of patients in each of the BMI categories who experienced an injection site-related AE were as follows: underweight (6.1%); normal weight (4.5%); overweight (5.1%); and obese (6.3%). BMI category was not significantly associated with percentage of injection site-related AEs , hydrocortisone was given to 1 patient with a mass, amoxicillin was given to 1 patient with an injection site reaction, sodium metamizole was given to 1 patient for pain, and betamethasone was given to 1 patient with a rash.Medications were provided when necessary for the treatment of injection site-related AEs, but in most cases  no concomitant medications were administered . However, concomitant medications The safety and efficacy of olanzapine LAI have been reported previously and have been shown to be similar to those of oral olanzapine -12, withp = .041). This may be related to unknown factors at the injection site that may make these patients at higher risk for both events and needs to be further explored. However, it should be noted that this relationship is based on a small number of patients with an event (n = 37) and that these shared risk factors imply only an association between the injection site-related AEs and PDSS and that these events are not necessarily causally related.Injection site-related AEs occurred in a small percentage of patients (5.3%). When these did occur, most were identified as mild (81.4%), with a median duration of 3 days. Pain was the most common type of event, occurring in 2.9% of patients, but each of the other types of injection site-related AEs occurred in fewer than 1% of patients. Dose volume and BMI did not appear to affect the probability of injection site-related AEs. The percentage of patients who experienced a PDSS event was greater among those who, at some point during the study, also experienced an injection site-related AE versus those who did not . In a very few instances, the pain resulted in the prescription of an analgesic. The other medications prescribed were antibiotics or an anti-inflammatory, prescribed for injection site reaction, mass, abscess, or rash. There were very few discontinuations due to injection site-related AEs.N = 170). In the second study (N = 53) injection site pain and injection site reaction were reported as AEs by 7.5% and 1.9% of patients, respectively. Thus, the 5.3% incidence of injection site-related AEs observed with olanzapine LAI appears within the same range as that observed with other injectable antipsychotics, despite the use of a larger needle size and a larger volume of medication being injected with olanzapine LAI.The incidence of injection site-related AEs in the present analysis was similar to that found in previous studies of injectable neuroleptics. In a recent review of a small number of olanzapine LAI studies, Cañas and Möller  noted anIt is not possible to avoid all local injection site-related AEs, but some may be prevented by proper injection techniques ,18. SomeThe authors recognize several limitations of this report and the studies from which the data were drawn. First, although the majority of the injection site-related AEs were injection site pain, none of the studies included a formal assessment of pain severity. Second, some patients reported injection site-related AEs as ongoing at the time of discontinuation from the study, and thus it is not possible to know the outcome of these events. Finally, our event-term search strategy required “injection site” to be specified in the event term; however there may be other events in the database which were related to the injection but were excluded because the location of the reaction was not specified.For patients treated with olanzapine LAI, injection site-related AEs were generally mild. Pain was the most common type of event, occurring in 2.9% of patients, but each of the other types of injection site-related AEs occurred in fewer than 1% of patients. The incidence and nature of these injection site events were generally similar to those occurring during treatment with other injectable antipsychotics. Importantly, the rate of discontinuation due to injection site-related AEs was low, suggesting that the risk of these adverse events will not change the benefit/risk profile for most patients considering treatment with depot medications.Ms. Atkins, Dr. Detke, Dr. McDonnell, Mr. Case, and Dr. Wang are employees and minor stockholders of Eli Lilly and Company.All of the authors participated in the drafting or critical review of this manuscript. MC and SW were primarily responsible for the statistical analysis. SA was primarily responsible for interpretation of one or more of the studies that were included in this report. HD and DM were primarily responsible for the planning and/or design of one or more of the studies that were included in this report. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-244X/14/7/prepub


These compounds display extreme selectivity and do not significantly inhibit the activity of 139 other kinases that were tested including ten AMPK family members. In all cell lines tested, WZ4003 and HTH-01-015 inhibit the phosphorylation of the only well-characterized substrate, MYPT1 (myosin phosphate-targeting subunit 1) that is phosphorylated by NUAK1 at Ser445. We also identify a mutation (A195T) that does not affect basal NUAK1 activity, but renders it ~50-fold resistant to both WZ4003 and HTH-01-015. Consistent with NUAK1 mediating the phosphorylation of MYPT1 we find that in cells overexpressing drug-resistant NUAK1[A195T], but not wild-type NUAK1, phosphorylation of MYPT1 at Ser445 is no longer suppressed by WZ4003 or HTH-01-015. We also demonstrate that administration of WZ4003 and HTH-01-015 to MEFs (mouse embryonic fibroblasts) significantly inhibits migration in a wound-healing assay to a similar extent as NUAK1-knockout. WZ4003 and HTH-01-015 also inhibit proliferation of MEFs to the same extent as NUAK1 knockout and U2OS cells to the same extent as NUAK1 shRNA knockdown. We find that WZ4003 and HTH-01-015 impaired the invasive potential of U2OS cells in a 3D cell invasion assay to the same extent as NUAK1 knockdown. The results of the present study indicate that WZ4003 and HTH-01-015 will serve as useful chemical probes to delineate the biological roles of the NUAK kinases.The related NUAK1 and NUAK2 are members of the AMPK (AMP-activated protein kinase) family of protein kinases that are activated by the LKB1 (liver kinase B1) tumour suppressor kinase. Recent work suggests they play important roles in regulating key biological processes including Myc-driven tumorigenesis, senescence, cell adhesion and neuronal polarity. In the present paper we describe the first highly specific protein kinase inhibitors of NUAK kinases namely WZ4003 and HTH-01-015. WZ4003 inhibits both NUAK isoforms (IC We describe the discovery of structurally diverse kinase inhibitors to dissect the physiological roles of the NUAK isoforms. We recommend use of an inhibitor-resistant NUAK1[A195T] mutant to verify that the physiological effects of these compounds is indeed mediated through inhibition of NUAKs The other members of the AMPK family activated by LKB1 include AMPKα isoforms, four MARK (microtubule-affinity-regulating kinase) isoforms, three SIK  isoforms, two BRSK (brain-specific kinase)/SAD isoforms and SNRK (SNF-related kinase), a testis-specific enzyme  and the closely related NUAK2 [SNARK (SNF1/AMPK-related kinase)] are members of the AMPK (AMP-activated protein kinase) family of protein kinases that are activated by the LKB1 (liver kinase B1) tumour suppressor protein kinase ATP was from PerkinElmer. Protein G–Sepharose, glutathione–Sepharose and an ECL kit was from GE Healthcare. P81 phosphocellulose paper was from Whatman. Doxycycline, DMSO, BSA and benzamidine were from Sigma–Aldrich. PMSF was from Melford. Novex 4–12% polyacrylamide Bis-Tris gels, LDS sample buffer, puromycin, hygromycin, blasticidin, PBS-EDTA-based Cell Dissociation Buffer and other tissue culture reagents were from Invitrogen Life Technologies. Instant Blue Coomassie stain was from Expedeon. PEI (polyethylenimine) was from Polysciences, and 1 M magnesium acetate solution was from Fluka.The Sakamototide substrate peptide (ALNRTSSDSALHRRR) was used as the NUAK1 and NUAK2 substrate in kinase assays . [γ-32P]445) , anti-MYPT1  and anti-NUAK1 . Antibody production was carried out under UK Home Office approved guidelines. The commercial antibodies used in the present paper are anti-ACC (acetyl-CoA carboxylase) , anti-(ACC p-Ser79) , anti-HA (haemagglutinin)–peroxidase (3F10)  and all HRP (horseradish peroxidase)-conjugated secondary antibodies were obtained from Thermo Scientific.The following antibodies were raised in sheep and affinity-purified on the appropriate antigen: anti-, using DYEnamic ET terminator chemistry  on Applied Biosystems automated DNA sequencers.All recombinant DNA procedures, electrophoresis, immunoblotting, immunoprecipitation and tissue culture were performed using standard protocols. NUAK1[A195T] mutagenesis was performed using the QuikChange® site-directed mutagenesis method (Stratagene) with KOD polymerase (Novagen). DNA constructs used for transfection were purified from +/+ and NUAK1−/− MEFs were cultured in DMEM supplemented with 10% (v/v) FBS and 2 mM glutamine, 1×antibacterial/antimycotic solution, 1% (v/v) non-essential amino acids and 1% (v/v) sodium pyruvate. HEK-293 Flp/In T-Rex cell lines were cultured in DMEM supplemented with 10% (v/v) FBS and 2 mM glutamine, 1×antibacterial/antimycotic solution, 100 μg/ml hygromycin and 15 μg/ml blasticidin. Supplementing the culture medium with 0.1 μg/ml doxycycline for 16–24 h induced protein expression in the HEK-293 Flp/In T-Rex cells. Cell counting was carried out using Invitrogen Countess following the manufacturer's protocol. A cell-detachment assay was carried out on HEK-293 cells using PBS-EDTA-based cell dissociation buffer as described previously ATP (450–500 c.p.m./pmol) and the indicated concentrations of inhibitors dissolved in DMSO. After incubation for 30 min at 30°C, reactions were terminated by adding 25 mM  EDTA to chelate the magnesium. Then, 40 μl of the reaction mix was spotted on to P81 paper and immersed in 50 mM orthophosphoric acid. Samples were washed three times in 50 mM orthophosphoric acid followed by a single acetone rinse and air drying. The incorporation of [γ-32P]ATP into Sakamototide was quantified by Cerenkov counting. The values were expressed as a percentage of the DMSO control. IC50 curves were developed and IC50 values were calculated using GraphPad Prism software.Active GST–NUAK1, GST–NUAK1[A195T] and GST–NUAK2 enzymes were purified using glutathione–Sepharose from HEK-293 cell lysates 36–48 h following the transient transfection of pEBG2T mammalian constructs expressing N-terminal GST-tagged NUAK1, NUAK1[A195T] or NUAK2. For peptide kinase assays, 96-well plates were used, and each reaction was performed in triplicate. Each reaction was set up in a total volume of 50 μl containing 100 ng of NUAK1 (wild-type or A195T mutant) or NUAK2 in 50 mM Tris/HCl (pH 7.5), 0.1 mM EGTA, 10 mM magnesium acetate, 200 μM Sakamototide, 0.1 mM [γ-In vitro activities of purified GST–NUAK1 and GST–NUAK1[A195T] were measured using Cerenkov counting of incorporation of radioactive 32P from [γ-32P]ATP into Sakamototide substrate peptide as described previously [32P]ATP into Sakamototide was quantified by Cerenkov counting. One unit of activity was defined as that which catalysed the incorporation of 1 nmol of [32P]phosphate into the substrate over 1 h.eviously . Reactio2, the insert was removed and the migration of cells into the 500 μm gap between the chambers was observed. The wound-gap healing properties of the cells were observed over a period of 15–20 h under a Nikon Eclipse Ti microscope with images taken every 2 min by a Photometrics cascade II CCD (charge-coupled device) camera using Nikon NIS Elements software.MEFs were split and an approximately equal number of cells were loaded into the left and right chambers of the IBIDI Self-Insertion Inserts . Each insert was placed in one well of a 12-well plate and the cells were seeded with or without treatment with the inhibitors. For the comparison of the migration properties of different MEFs on the same video, a single insert was used and an equal number of MEFs were counted and loaded on either chamber of the same insert. To study the effect of inhibitors on cell migration, wound-healing assays on MEFs were also carried out on separate inserts with or without treatment with a 10 μM concentration of WZ4003 or HTH-01-015. Inhibitors were added to the cells 1 h before the start of the migration assay. The experiments were carried out in triplicate. After overnight incubation at 37°C and 5% COCell proliferation assays were carried out colorimetrically in 96-well plates using the CellTiter 96® AQueous Non-Radioactive Cell Proliferation Assay kit (Promega) following the manufacturer's protocol. Initially, 2000 cells per well were seeded for U2OS cells and 3000 cells per well were seeded for MEFs. The proliferation assays were carried out over 5 days in the presence or absence of 10 μM HTH-01-015 or WZ4003.5 cells suspended in DMEM containing 1% (w/v) BSA were added to the upper chambers in triplicate and chemoattractant [DMEM containing 10% (v/v) FBS] was added to the lower wells. The chambers were kept at 37°C in 5% CO2 for 16 h in the presence or absence of 10 μM HTH-01-015 or WZ4003 both in the upper and lower wells. Non-invaded cells were removed from the upper face of the filters by scraping, and cells that had migrated to the lower face of the filters were fixed and stained with Reastain Quick-Diff kit (Reagena) and images (×10 magnification) were captured. For cell invasion assays, statistical significance was assessed using GraphPad Prism 5.0.The ability of U2OS cells to invade in the presence or absence of 10 μM HTH-01-015 or WZ4003 was tested in a growth-factor-reduced Matrigel™ invasion chamber  as described previously . Cells whttp://www.kinase-screen.mrc.ac.uk/) against a panel of 140 protein kinases as described previously , we observed that even at very high concentrations of 30 μM, WZ4003  and homozygous NUAK1-knockout (NUAK1−/−) MEFs using a 2D wound-healing assay. Consistent with NUAK1−/− MEFs being more adhesive, we found that they migrated slower than wild-type cells and presented a more ‘flattened’ adherent phenotype . This phenotype could be largely rescued by retroviral overexpression of NUAK1+/+ into NUAK1−/− MEFs (Supplementary Movie S2 at http://www.biochemj.org/bj/457/bj4570215add.htm). We next investigated whether the WZ4003 and HTH-01-015 inhibitors could inhibit cell migration and observed that treatment of NUAK1+/+ MEFs with 10 μM WZ4003 or HTH-01-015 markedly reduced cell migration in the wound-healing assay (Previous work suggested that RNAi-mediated knock down of NUAK1 promoted cell adhesion , which whenotype A. A moving assay B.Previous studies have suggested that inhibiting NUAK1 would suppress proliferation . We therPrevious work has implicated NUAK1 in controlling the invasive ability of various cell types –13. To tin vivo, we observe that these compounds inhibited MYPT1 Ser445 phosphorylation as well as cell migration, invasion and proliferation to a similar extent as knock out in MEFs or knock down in U2OS cells of NUAK1. The identification of the A195T mutation that renders NUAK1 ~50-fold resistant to WZ4003 and HTH-01-015 also provides an important approach to validate that biological effects of these compounds are indeed mediated through inhibition of NUAK1 rather than through an off-target effect. Although as a proof of concept, we have shown that overexpression of the NUAK1[A195T] mutant, but not wild-type NUAK1, renders MYPT1 phosphorylation resistant to WZ4003 and HTH-01-015, this approach is not ideal, as the overexpression of NUAK1 has the potential to have an impact on biological processes by inducing non-physiological phosphorylation of cellular proteins. In future work we would recommend that gene-editing technologies be deployed to generate an endogenous NUAK1[A195T] knock-in mutation. Such knock-in cell lines should be rendered greatly resistant to the WZ4003 and HTH-01-015 inhibitors and therefore any effects that these compounds have that is mediated through inhibition of NUAKs should be suppressed by this mutation.WZ4003 and HTH-01-015 are remarkably selective NUAK kinase inhibitors, and do not significantly inhibit the activity of any of the 139 other protein kinases we have investigated  and 2. C50 values of the WZ4003 and HTH-01-015 compounds for inhibiting NUAK1 are in the range 20–100 nM when assayed at 0.1 mM ATP in vitro. On the basis of the structures of these compounds, it is likely that they are acting as ATP-competitive inhibitors. As concentrations of ATP in cells are over 20-fold higher than our in vitro assays, this is likely to account for why relatively high concentrations of 3–10 μM WZ4003 and HTH-01-015 are required to maximally suppress MYPT1 Ser445 phosphorylation in vivo. We have devoted considerable effort to generate more potent NUAK1 inhibitors and have indeed identified two analogues of HTH-01-015, namely XMD-17-51 and XMD-18-42, that inhibit NUAK1 with greater potency. However, these compounds suffer from the drawback that they are less selective than WZ4003 and HTH-01-015 and inhibit other kinases implicated in controlling cell growth and proliferation (The ICferation  and 4. Xferation .in vivo. Therefore comparing the effects of WZ4003 with NUAK1-selective inhibitors could provide insights into the relative contributions of NUAK isoforms in mediating physiological processes. In vitro NUAK1 and NUAK2 are equally efficient at phosphorylating MYPT1 at Ser445 and both isoforms interact similarly with the MYPT1–PP1 complex  mutants be deployed to ensure any biological effects arising from NUAK1 inhibitors is indeed desensitized by this mutation. In future studies it will be exciting to establish the effects that WZ4003 and HTH-01-015 have on biological process recently proposed to be controlled by NUAK isoforms, such as Myc-driven tumour cells 5T mutant and mela


Without affecting cytotoxicity, both coumarines tested were effective inhibitors/scavengers of ROS produced by neutrophils on extracellular level. HHC markedly diminished oxidant production and also, intracellularly, decreased PKC activity and partly phosphorylation of PKCα, βII. On the other hand, we did not observe any effect of coumarin derivatives on phosphorylation of PKCδ and on phosphorylation of the NADPH oxidase subunit p40phox, which were suggested to be involved in the PMA-dependent intracellular activation process. In agreement with our previous findings, we assume that the different molecular structures of HHC and HHDC with their different physicochemical and free radical scavenging characteristics are responsible for their diverse effects on the parameters tested. To specify the site of action of the synthetic coumarin derivatives 7-hydroxy-3-(4′-hydroxyphenyl) coumarin (HHC) and 7-hydroxy-3-(4′-hydroxyphenyl) dihydrocoumarin (HHDC), we evaluated their effects on extra- and intracellular reactive oxygen species (ROS) formation in phorbol-myristate-13-acetate (PMA) stimulated human neutrophils. We studied also the effects of HHC and HHDC on possible molecular mechanisms which participate in the activation of NADPH oxidase, that is, on PKC activity, on phosphorylation of some PKC isoforms ( Neutrophils are key cells of the first line of defense, but they are also considered potent inflammatory cells causing tissue damage. Thus the ability of compounds which prevent extensive and potentially dangerous activation of neutrophils has been proposed as an important injury-limiting way. Coumarins belong to the group of plant-derived polyphenolic compounds possessing broad biochemical and pharmacological effects, like anti-HIV, anti-inflammatory, antioxidant, antibacterial, anticoagulant, and anticancer activities –4. Over phox, p47phox, p67phox, and Rac2) migrate to the membranes, where they associate with the membrane-bound component (flavocytochrome b558) to assemble the catalytically active NADPH oxidase, resulting in the delivery of ROS into the extracellular environment or inside into the phagocytic vesicle , 0.085 mmol/L aprotinin, 1.53 mmol/L bestatin hydrochloride, 1.40 mmol/L E-64 [N-(trans-epoxysuccinyl)-L-leucine 4-guanidinobutylamide], 1.90 mmol/L leupeptin hemisulfate salt, and 4.22 mmol/L pepstatin in DMSO, were purchased from Sigma-Aldrich Chemie , horse radish peroxidase (HRP) and catalase from Merck , and Lymphoprep (density 1.077 g/mL) from Nycomed Pharma AS .HHC and HHDC were kindly provided by Dr. Juraj Harmatha from the Institute of Organic Chemistry and Biochemistry, AV ČR, Prague, Czech Republic, and Professor Jan Šmidrkal from the Institute of Chemical Technology, Department of Dairy and Fat Technology, AV ČR, Prague, Czech Republic. μL 1 NaOH and 980 μL Tyrode solution . The stock solution (5 mmol/L) was further diluted with Tyrode solution to give HHC samples, concentrations 0.01–100 μmol/L. The corresponding final concentrations of NaOH were 0.4–400 μmol/L; at these concentrations, the solvent agent alone did not reduce the activity and viability of neutrophils.HHC (1.27 mg) was dissolved in a mixture of 20 All other chemicals of analytical grade were from available commercial sources. This work was approved by the Local Ethic Committee, Institute of Experimental Pharmacology and Toxicology SAS.2HPO4, 1.5 KH2PO4, and pH 7.4, at final concentration of 1 × 107 cells/mL. The obtained cell suspension contained more than 96% viable cells, as evaluated by trypan blue exclusion [2HPO4, 1.5 KH2PO4, 1.8 CaCl2, 0.5 MgCl2 · 6H2O, and pH 7.4.Fresh blood was taken at the blood bank from healthy volunteers  by antecubital venipuncture and was immediately mixed with 3.8% v/w trisodium citrate, in the ratio of 9 mL of blood to 1 mL of citrate, in polypropylene centrifugation tubes. Erythrocytes were allowed to sediment in 3% dextran solution  and neutrophils were separated by gradient centrifugation on Lymphoprep . After hypotonic lysis of contaminating erythrocytes, neutrophils were washed and resuspended in calcium- and magnesium-free phosphate buffer saline in mmol/L: 137 NaCl, 2.7 KCl, 8.1 Naxclusion , 25. Priμmol/L) on extra- and intracellular ROS production was measured in unstimulated and PMA (0.05 μmol/L) stimulated neutrophils (5 × 105/sample) by isoluminol-/luminol-enhanced chemiluminescence (CL). Extracellular CL was determined in the system containing isoluminol (5 μmol/L) and HRP (8 U/mL). Intracellular CL was measured with luminol (5 μmol/L) in the presence of the extracellular scavengers superoxide dismutase (100 U/mL) and catalase (2 000 U/mL). CL was evaluated in a microplate luminometer Immunotech LM-01T (Czech Republic) at 37°C. Data were based on integral values of CL over 1800 s,  [The effect of HHC or HHDC  , 27.μL samples consisted of 50 μL aliquots: HRP (2 U/mL), luminol (10 μmol/L), HHC, or HHDC solutions in the concentrations of 1, 10, or 100 μmnol/L. The reaction was initiated by adding hydrogen peroxide at the final concentration of 100 μmol/L. The chemiluminescence responses were measured for 10 minutes at 37°C in a microplate luminometer Immunotech LM-01T (Czech Republic).In the cell-free system, luminol, horseradish peroxidase, and hydrogen peroxide were used for chemiluminescence generation . The 2005/mL) were incubated 30 min with HHC  or HHDC  at 37°C. Neutrophils were then stimulated with PMA  at 37°C for 3 min. The reaction was stopped by addition of 10 vol of ice-cold PBS. After centrifugation  the cells were resuspended in sample buffer , sonicated on ice, and centrifuged . The cytosolic fractions were transferred to a prechilled 1.5 mL microcentrifuge tube and stored at –70°C. Protein content of cytosol fraction was measured using Bradford Dye Reagent Detection Kit . PKC activity was measured with a Nonradioactive Protein Kinase Assay kit , which is based on a solid-phase enzyme-linked immunoabsorbent assay (ELISA), utilising a specific synthetic peptide as substrate for PKC and a polyclonal antibody recognizing the phosphorylated form of the substrate. The assay is developed with tetramethylbenzidine substrate and a colour develops in proportion to PKC phosphotransferase activity. The colour development was stopped with acid stop solution and the intensity of the colour was measured in a microplate reader at 450 nm. The data were expressed as relative kinase activity per 1 mg of protein. PKC activity in the cytosol was detected by the modified method of Varga et al. . Isolateα, βII, and δ, as well as NADPH oxidase subunit p40phox, was detected using the method described by Jančinová et al. [Phosphorylation of PKC isoenzymes á et al. , 27.μL) containing 5 × 106 cells was preincubated at 37°C for 60 seconds with different concentrations of HHC or HHDC  prior to addition of PMA (0.15 μmol/L). Incubation with PMA (5 min) was stopped by using a solubilisation buffer . The suspension was sonicated at 4°C for 20 minutes and centrifuged at 14 000 ×g at 4°C for 5 min to remove unbroken cells. The supernatant was taken for measuring total protein by using Bradford Dye Reagent Detection Kit from Bio-Rad and for blotting assay. The supernatant for blotting assay was boiled for 5 min with sample buffer  and samples were loaded on 9.8% SDS polyacrylamide gels. Proteins (20 μg per lane) were separated by electrophoresis and transferred to Immobilon-P Transfer Membrane . From the two strips taken, one was detected for PKCα, βII (area between 60 and 100 kD) and the second for detection of β-actin (30–60 kD), which represented the internal control. Membrane strips were blocked for 60 min with 1% bovine serum albumin in Tris buffered saline . This was then followed by 60 min incubation in the presence of the primary antibodies: anti-phospho-PKC-α, βII (Thr638/641) (1 : 5 000) or β-actin  . The membranes were subsequently washed six times with TBS and incubated 60 min with the secondary antibody conjugated to horseradish peroxidase , and the activity of horseradish peroxidase of the bands corresponding to individual PKC isoforms was visualised using Enhanced Chemiluminescence Western Blotting Detection Reagents .The suspension of isolated neutrophils  for 15 min and washed overnight in diluent buffer . This was followed by 60 min incubation in the presence of primary antibodies: anti-phospho-p40phox (T154) (1 : 5 000) or PKCδ (Thr505) (1 : 1 000) . The membranes were subsequently washed six times with TBS and incubated 60 min with the secondary antibody conjugated to horseradish peroxidase , and the activity of horseradish peroxidase of the bands corresponding to the individual NADPH oxidase subunit or to PKC isoform was visualised using Enhanced Chemiluminescence Western Blotting Detection Reagents . Autoradiogram bands were quantified using the Image J programme. The optical density of each PKC or NADPH oxidase band was corrected by the optical density of the corresponding β-actin band [NADPH oxidase subunit p40tin band , 27.μL; 30 000 cells/sample) and 20 μL of Tyrode's solution were incubated with 50 μL of HHC or HHDC (1–100 μmol/L) for 15 min at 37°C. Ten microliters of a mixture of luciferin (1.6 μg/sample) and luciferase (45 000 U/sample) was added, and the chemiluminescence was recorded for 60 s. In each experiment, the chemiluminescence of ATP standards (1–500 nmol/L) was measured and the concentrations of ATP in samples were calculated from the calibration curve. The total ATP content was assessed immediately after sonication of neutrophils for 10 s [The cytotoxic effect of HHC and HHDC was evaluated by means of ATP liberation by the luciferin-luciferase chemiluminescence. The neutrophil suspension , representing 7.6% of the total ATP content (158.7 ± 12.1 nmol/L ATP), as determined immediately after complete neutrophil destruction. The results indicate that treatment with increasing concentrations of HHC and HHDC did not cause neutrophil damage. To determine cytotoxicity of HHC and HHDC, we tested their effect on ATP liberation . In the For activation of isolated human neutrophils, the soluble stimulus PMA was used, which bypasses receptors and activates NADPH oxidase via redistribution of PKC and phosphorylation of several proteins. As mentioned above, PMA is useful in investigating signal transduction pathways leading to NADPH-oxidase activation in plasma (extracellular) and granule membranes (intracellular) , 11.  Fiμmol/L, decreased significantly extracellular ROS production, as measured by chemiluminescence. The concentrations of HHC and HHDC producing 50% inhibition (IC50) of control extracellular chemiluminescence were 1.04 ± 0.20 μmol/L and 1.01 ± 0.13 μmol/L, respectively (ectively . μmol/L decreased significantly intracellular ROS production (IC50: 5.24 ± 0.57 μmol/L), HHDC was slightly effective only in the highest concentration tested  and HHDC (IC50: 2.47 ± 0.04)  . α, βII, and δ, which are involved in the oxidative burst as key activators of NADPH oxidase. First we analysed whether modulation of PKC activity by HHC or HHDC might mediate their inhibitory effect on PMA stimulated ROS production. HHC in the concentrations of 1, 10, and 100 μmol/L significantly decreased PKC activity to 87.7 ± 4.2%, 74.8 ± 6.3%, and 46.3 ± 1.8%, respectively. On the other hand, HHDC did not significantly influence PKC activity , we further specified the effect of HHC and HHDC on phosphorylation of PKCα, βII, and δ.Stimulation of neutrophils with phorbol esters activates the PKC isoforms activity . Since tα, βII, and δ in comparison with unstimulated human neutrophils. Treatment with HHC or HHDC in the concentrations of 10 and 100 μmol/L resulted in decrease in the level of PKCα, βII as compared to those of control but did not affect phosphorylation of PKCδ (Figures After PMA stimulation, we observed increased phosphorylation of PKC Figures  and 7. phox in intracellular ROS production was reported [phox subunit in PMA stimulated human neutrophils. As shown in phox subunit, yet HHC and HHDC failed to influence it.Recently, the selective role for p40reported , 13. We  Regulation of neutrophil activity is one of the important factors in achieving host defense and avoiding tissue-damaging inflammation. Recently, great attention has been devoted to polyphenols of natural origin as well as to their synthetic derivatives. Many of them were found to possess effects which could be used in prevention and support therapy of chronic inflammatory diseases –35. Prev Our results showed that neutrophils activated by PMA responded by an oxidant production composed of both extra- and intracellular component of chemiluminescence. We found the extracellular ROS production to be predominant, as also demonstrated by Björnsdottir et al.  and DahlThe effective inhibition of cell-free system chemiluminescence by both compounds tested  showed tin vitro and in vivo [ The relationships between antioxidant activities and chemical structures of coumarin derivatives are important factors which may influence the oxidant production  in vivo , 40–42. Polyphenols were reported to affect cell functions by modifying plasma membrane structure and physical characteristics, such as fluidity and electrical properties. These effects can be observed both when polyphenols are adsorbed on the membrane and when they are inserted into the bilayer . The low In biological systems, ROS are generated by a number of enzymatic systems, and modifications of plasma membrane structure can result in functional changes, including the activity of membrane-associated enzymes and the modulation of signal transduction , 43. α, βII phosphorylation. Inhibition of PKC activity and PKCα, βII phosphorylation by HHC only, indicates that the presence of the double bond between C3 and C4 at ring C is necessary for this effect. Structure dependence of the inhibitory effect of polyphenolic antioxidants on signal transduction enzymes, such as PKC, was found also by Varga et al. [δ in the PMA-dependent intracellular activation process was reported [δ. Inhibition of PKC or downregulation of its intracellular expression and activity has been proposed as an important mechanism of the antioxidant effect of polyphenols , 44, 45.a et al. . Althougreported , we did phox, which is specifically translocated to intracellular phagosomal and granule membranes, was also indicated as a further determining factor for intracellular ROS production [phox phosphorylation by PKC inhibitors was found by Bouin et al. [phox. One of the cytosolic components of the NADPH oxidase, p40oduction , 12. Morn et al.  and Somen et al. . Our resWe extended our previous findings about the actions of the coumarine derivatives HHC and HHDC on the activity of human neutrophils by investigating the influence of extra- and intracellular ROS formation and some possible molecular mechanisms affecting the regulation of oxidant production. The presented results suggest that, in pathological processes in which neutrophils are involved, HHDC may act predominantly as a potent inhibitor/scavenger of extracellularly produced ROS. HHC may act both extra- and intracellularly, and, besides its direct interference with ROS, it may interfere also with the PKC signalling pathway. These findings confirmed our previous assumption that the different effects of the coumarine derivatives tested might be due to their diverse molecular structures, which provides them with different physicochemical and free radical scavenging characteristics.


The PDGF signaling pathway plays a major role in several biological systems, including vascular remodeling that occurs following percutaneous transluminal coronary angioplasty. Recent studies have shown that the LDL receptor-related protein 1 (LRP1) is a physiological regulator of the PDGF signaling pathway. The underlying mechanistic details of how this regulation occurs have yet to be resolved. Activation of the PDGF receptor β (PDGFRβ) leads to tyrosine phosphorylation of the LRP1 cytoplasmic domain within endosomes and generates an LRP1 molecule with increased affinity for adaptor proteins such as SHP-2 that are involved in signaling pathways. SHP-2 is a protein tyrosine phosphatase that positively regulates the PDGFRβ pathway, and is required for PDGF-mediated chemotaxis. We investigated the possibility that LRP1 may regulate the PDGFRβ signaling pathway by binding SHP-2 and competing with the PDGFRβ for this molecule.To quantify the interaction between SHP-2 and phosphorylated forms of the LRP1 intracellular domain, we utilized an ELISA with purified recombinant proteins. These studies revealed high affinity binding of SHP-2 to phosphorylated forms of both LRP1 intracellular domain and the PDGFRβ kinase domain. By employing the well characterized dynamin inhibitor, dynasore, we established that PDGF-induced SHP-2 phosphorylation primarily occurs within endosomal compartments, the same compartments in which LRP1 is tyrosine phosphorylated by activated PDGFRβ. Immunofluorescence studies revealed colocalization of LRP1 and phospho-SHP-2 following PDGF stimulation of fibroblasts. To define the contribution of LRP1 to SHP-2-mediated PDGF chemotaxis, we employed fibroblasts expressing LRP1 and deficient in LRP1 and a specific SHP-2 inhibitor, NSC-87877. Our results reveal that LRP1 modulates SHP-2-mediated PDGF-mediated chemotaxis.Our data demonstrate that phosphorylated forms of LRP1 and PDGFRβ compete for SHP-2 binding, and that expression of LRP1 attenuates SHP-2-mediated PDGF signaling events. Pdgfb or Pdgfrb in mice leads to an almost complete lack of pericytes in certain vascular beds Despite significant advances in the treatment of severe coronary artery blockage, restenosis continues to represent a serious clinical problem by impeding long-term success of vascular interventions in vivo and in vitro studies reveal that the LDL receptor-related protein 1 (LRP1) is a physiological modulator of the PDGF signaling pathway. LRP1 is abundantly expressed in vascular SMC, and is a large endocytic and signaling receptor that mediates the endocytosis and subsequent degradation of several ligands including apoE-rich lipoproteins, proteases, and protease-inhibitor complexes Lrp1 gene in vascular SMC (smLRP1−/−) on a background of LDL receptor deficiency, causes smooth muscle cell proliferation, aneurysm formation, and a significant increase in susceptibility to cholesterol-induced atherosclerosis Both The mechanisms by which LRP1 modulates the PDGF signaling pathway are not well understood. Tight regulation of the PDGFRβ is critical, as excessive activation induces tumor formation SHP-2 is a ubiquitously expressed, cytoplasmic protein tyrosine phosphatase (PTP) that contains two SH2 domains Since phosphorylated forms of LRP1 and the PDGFRβ are both capable of binding SHP-2, we hypothesized that these two receptors may compete for SHP-2 binding, and if so, we reasoned that this may represent one mechanism by which LRP1 suppresses PDGFRβ-mediated signaling. Our results reveal that phosphorylated forms of the LRP1 cytoplasmic domain and the PDGFRβ kinase domain (KD) bind with very high affinity to SHP-2, and compete with one another for this interaction. Further, we observed that SHP-2 co-immunoprecipitates and colocalizes with LRP1 following PDGF stimulation of cells, and that LRP1 attenuates SHP-2 mediated cell migration. Taken together, the data reveal a critical role for LRP1 in modulating SHP-2-mediated PDGF signaling events.−/− (PEA-13) and B41 clones (LRP1−/− deficient cells transfected with human LRP1) have been described S-transferase as described 2. Soluble PDGFRβ ectodomain (sPDGFr) was purchased from Sino Biological, Inc.Newborn rat kidney (NRK) fibroblasts, human WI-38 fibroblasts and murine aortic smooth muscle (MOVAS) cells were purchased from ATCC. LRP1For pull-down experiments, NRK fibroblasts were grown to 70% confluence on 60 mm dishes. Cells were lysed in lysis buffer , 2 mM activated sodium orthovanadate, 1 mM PMSF, 0.3 µM okadaic acid, protease inhibitor cocktail tablets (Roche Diagnostics), in TBS) and cell lysates were incubated with phosphorylated or unphosphorylated GST:LRP1-ICD immobilized on Glutathione-Sepharose. Following incubation and washing, proteins were eluted and separated by SDS-PAGE and analyzed by immunoblot analysis using the indicated antibodies.Prior to lysis, cells were incubated for 30 minutes on ice with the cell-permeable crosslinking agent, DSP (dithiobis[succinimidylpropionate]) . Following incubation, cells were washed and then collected in lysis buffer , 2 mM activated sodium orthovanadate, 1 mM PMSF, 0.3 µM okadaic acid, protease inhibitor cocktail tablets (Roche Diagnostics), in TBS). Lysates were incubated with Dynabead Protein G  overnight at 4°C. Immunoprecipitates were washed, separated by SDS-PAGE on 4–12% tris-glycine precast gels (Invitrogen), and transferred to nitrocellulose membranes for immunoblot analysis. Whole cell lysates were separated by SDS-PAGE on 4–12% tris-glycine precast gels (Invitrogen) and then electrophoretically transferred to nitrocellulose membranes. Immunoblots were first incubated for 1 hour at room temperature in buffer containing 50 mM Tris, 150 mM NaCl, 0.1% Tween 20, and 5% nonfat dry milk. The membranes were then incubated overnight with specific antibodies and washed in buffer containing 50 mM Tris, 150 mM NaCl, and 0.1% Tween 20. Antibody binding to the immunoblots was detected by incubation with an appropriate IRDye® (LI-COR Biosciences) -conjugated secondary antibody. Immunoreactive bands were detected using LI-COR Odyssey Infrared Imaging System.maxA = A/(1+ dK/[L])+B, where A = absorbance, Amax = maximum absorbance, Kd = dissociation constant, L = ligand concentration and B = background absorbance.Immulon 4HB microtiter plate wells were coated overnight at 4°C with SHP-2 (5 µg/mL) in TBS, pH 8 (coating buffer). The wells were then blocked with 5% BSA  in TBS, pH 8 for 1 h at room temperature. After washing the wells with TBS containing 0.05% Tween 20, the indicated concentrations of active PDGFRβ kinase domain (KD), sPDGFr or GST:LRP1-ICD were added to the wells in incubation buffer , and incubated overnight at 4°C. Bound proteins were detected by addition of anti-GST HRP-conjugated antibody  or anti-PDGFRβ Rabbit mAb  followed by Goat anti-rabbit HRP-conjugated . TMB microwell peroxidase substrate  was added to the wells and the amount of bound ligand was measured spectrophotometrically at 620 nm. Data were analyzed by nonlinear regression analysis using Graphpad Prism 5 software employing the equation: 2. The substrate for SHP-2 was Src pY529 (TSTEPQ-pY-QPGENL), (Upstate biotech) and was used at a concentration of 250 µM. 25 uL of each sample were transferred to a 96 well Immulon 4HB microtiter plate, in duplicate. TBS buffer with 1 mM ATP and 1 mM MgCl2 with indicated proteins were assayed as a control. Malachite green solution (Echelon Biosciences) was added to each well and allowed to incubate for 15 minutes at room temperature. Liberated phosphate complexed with malachite green forms a colored complex. Absorbance was spectrophotometrically measured at 620 nm.The catalytic activity of SHP-2 was assessed using a malachite green phosphatase assay kit (Echelon Biosciences). Recombinant SHP-2 (400 nM) was incubated with PDGFR KD (400 nM) or sPDGFRr (400 nM) or GST-LRP1-ICD (400 nM) in the presence of 1 mM ATP and 1 mM MgClBinding of GST:LRP1-ICD to recombinant human SHP-2  was measured using a BIA 3000 optical biosensor . For these studies, a CM5 BIAcore sensor chip was activated, and GST:LRP1-ICD (either phosphorylated or unphosphorylated) was coupled as described Murine aortic SMC (MOVAS) were grown in 6-well plates in DMEM supplemented with 10% fetal bovine serum. Cells were serum-starved for 24 h before the experiment. To block dynamin-mediated endocytosis, cells were treated with a small molecule inhibitor of dynamin, dynasore WI-38 fibroblasts were serum starved overnight and then stimulated with PDGF-BB (30 nm/mL). Cells were serum-starved for 24 hours before adding PDGF-BB (30 ng/mL). The cells were incubated at 37°C for 10 min followed by washing in PBS and fixation in 3.7% formaldehyde, 5% sucrose in PBS for 20 minutes at room temperature. The fixed cells were permeabilized with 0.5% Triton X-100 for 5 minutes, blocked with 5% Donkey Serum for 1 hour, and then incubated with anti-phospho SHP-2 rabbit monoclonal (Santa Cruz) and mouse monoclonal 8G1 anti-LRP IgG (5 µg/mL) for overnight at 4°C. Slides were then washed and incubated with donkey Alexa-conjugated anti-rabbit and anti-mouse secondary antibodies (Invitrogen) for 1 h at room temperature. Stained coverslips were washed and mounted onto glass slides using FluorSave Reagent  and viewed with laser scanning system Radiance 2100 (Zeiss/Bio-Rad) equipped with the argon/krypton (488/568 nm) and red diode (638 nm) lasers. The images were acquired and stored in RAW format using LaserSharp2000 software . The images were exported into TIFF format and processed by Adobe Photoshop software (Adobe Systems) for publication. Adjustments of brightness and contrast were made for the entire image and were performed identically for both “single channel” and “merged” images.−/−) and B41 clones (LRP1+/+) were seeded 2×104 cells per filter onto the insert membrane and grown overnight. Cells were pretreated for 3 hours with 30 µM SHP-2 inhibitor, NSC-87877 . Either buffer or PDGF-BB (30 ng/ml) was applied to the bottom chamber and incubation was carried out for 4 hours at 37°C. The topsides of filters were cleared of cells with a cotton swab. Cells were fixed with ice-cold methanol for 5 min and nuclei were stained with Vectashield mounting medium with Dapi (Vector Laboratories). Filters were mounted onto glass slides and fluorescent images of three random fields from two independent experimental membranes for each experimental condition were acquired on a Nikon E800 Eclipse microscope and quantified using software Volocity (Improvision).Migration experiments were performed with 24-well plates containing 5-µm pore size transwell filters (Costar). PEA-13  by Src-family kinase members as well as the activated PDGFRβ D = 18+/−4 nM). In contrast, the unphosphorylated form of GST:LRP1-ICD failed to bind to SHP-2. As a control experiment, the binding of GST to SHP-2 immobilized on microtiter wells was also performed, and no binding was detected (data not shown). An additional control for this experiment measured the binding of GST:LRP1-ICD to BSA-coated wells, which confirmed no binding to BSA. Since ELISA based experiments measure high affinity interactions, to determine if unphosphorylated forms of the LRP1-ICD interacts weakly with SHP-2, we performed surface plasmon resonance experiments in which phosphorylated or unphosphorylated GST:LRP1-ICD was bound to SPR chips and SHP-2 was injected over these surfaces. The results of this study verified the interaction between SHP-2 and the phosphorylated form of GST:LRP1-ICD . In contrast, a relatively weak interaction of non-phosphorylated forms of the PDGFRβ KD with immobilized SHP-2 was detected (KD = 326+/−68 nM).A specific interaction between SHP-2 and phosphorylated tyrosines 763 and 1009 in the cytoplasmic domain of the activated PDGFRβ has been demonstrated by co-immunoprecipitation analysis Since phosphorylated forms of LRP1 and PDGFRβ KD both bind to SHP-2 with high affinity, we conducted experiments to determine if these two receptors compete for the binding of SHP-2. To investigate this possibility, we incubated microtiter wells coated with SHP-2 with increasing concentrations of phosphorylated GST:LRP1-ICD in the presence of constant amounts of phosphorylated PDGFRβ KD. The results of this experiment  demonstrSHP-2 has been reported to dephosphorylate the PDGFRβ at tyrosines 771, 751 and 740 To determine the effect of SHP-2 phosphorylation by the PDGFRβ KD on the catalytic potential of SHP-2, we measured the phosphatase activity of SHP-2 after incubating with the PDGFRβ kinase domain in the presence of ATP. As a control for this experiment, we also used the soluble ectodomain of the PDGFRβ (sPDGFr). The results  demonstrPrevious studies reveal that activation of the PDGFRβ results in tyrosine phosphorylation of LRP1 To examine the possible formation of a complex between LRP1 and phospho-SHP-2, we utilized WI38 fibroblasts which express abundant levels of LRP1 and SHP-2, thus making them an optimal cell system in which to investigate associations between these two molecules. Following PDGF stimulation, cells were chilled and incubated with the cell-permeable crosslinking agent, DSP (dithiobis[succinimidylpropionate]) prior to analysis. The results  reveal tSHP-2 is known to participate in PDGF-mediated chemotaxis, and to determine if LRP1 modulates the function of SHP-2 mediated chemotactic signaling, we examined the migration of LRP1-expressing (B41) and LRP1-deficient fibroblasts to PDGF-mediated chemotaxis. To examine the contribution of SHP-2 to this process, we employed the specific SHP-2 inhibitor, NSC-87877, which selectively inhibits the catalytic activity of SHP-2 in vivo observations in smLRP1−/− mice where increased levels of active PDGFRβ and increased PDGF-signaling are observed Excessive vascular smooth muscle cell growth and migration are hallmark features of many vascular occlusive diseases including atherosclerosis and restenosis after percutaneous transluminal angioplasty (PTCA) Activation of the PDGFRβ upon binding of its ligand leads to a transient tyrosine phosphorylation of the LRP1-ICD Based on the important role of SHP-2 in facilitating PDGF-mediated signaling events, we hypothesized that the association of SHP-2 with phosphorylated forms of LRP1 may attenuate PDGF signaling events. To test this hypothesis, we first conducted experiment to measure the affinity of SHP-2 for phosphorylated forms of the LRP1-ICD and compared this value to its affinity for activated forms of the PDGFRβ. These studies confirmed high affinity interactions between SHP-2 and the phosphorylated forms of the LRP1-ICD that were comparable to those measured for its interaction with the PDGFRβ. Additional experiments demonstrated a competition between these two receptors for SHP-2. We conclude from these experiments that phosphorylated forms of LRP1 binds SHP-2 with an affinity that is comparable to that of the PDGFRβ, and is thus capable of sequestering SHP-2 and preventing its association with activated forms of the PDGFRβ. Interestingly, we confirmed that LRP1 co-immunoprecipitates and co-localizes with SHP-2 in fibroblasts following stimulation with PDGF. This co-localization occurs most prominently in large vesicles, likely multivesicular bodies. It is tempting to speculate that LRP1 may bind and sequester SHP-2 in multivescular bodies. Of interest in this regard are the recent studies of Taelman et al. In summary, findings in the present study demonstrate that LRP1 modulates SHP-2-mediated PDGFRβ signaling events. This occurs following tyrosine-phosphorylation of LRP1 that is mediated by activated PDGFRβ. Elucidating mechanisms which modulate PDGFRβ signaling will give a greater understanding of the process of vascular smooth muscle remodeling in the context of cardiovascular disease, and possibly other PDGFR signaling-driven processes such as cancer and pulmonary fibrosis.


Ovomucoid (OM) has two carbohydrate chains on each of the first and second domains and one in the third. The contribution of the covalently bound carbohydrate chains to the overall OM allergenicity is controversial. Another aspect directly related with the immunological properties of OM that has not been studied in depth is the importance of the carbohydrate chains on its digestibility.The aim of the study was to assess the involvement of the carbohydrate moieties of OM in its digestibility and allergenic properties.IgE-binding and basophil activation by glycosylated and enzymatically deglycosylated OM (dOM) were compared using blood from egg-allergic patients. The peptides obtained after digestion using a physiologically relevant model were identified by RP-HPLC-MS/MS and the IgE-binding of the resulting fragments was evaluated by DOT-Blot.No structural changes were observed after deglycosylation of OM. 80% of the patients showed lower IgE binding to dOM as compared with OM and, in some patients, IgE reactivity could not be inhibited by pre-incubation with dOM. A subtle reduction in the percentage of activated basophils was observed when incubated with dOM as compared to OM. Following simulated digestion, dOM was more extensively degraded than OM, particularly during the gastric phase and both, OM and dOM, yielded, after the duodenal phase, immunoreactive fragments that were totally or partially coincident with previously described epitopes.&ClinicalRelevance: this work demonstrated an enhanced IgE reactivity towards carbohydrate containing OM in some egg-allergic patients that could be attributed to cross-sensitization or sensitization to the glycosylated components. The carbohydrate chains contributed to an increased resistance to proteolysis, and thus, to its allergenic potency. Evaluation of the products of digestion of OM and dOM revealed the presence of high-frequency IgE-binding epitopes that could remain linked by disulphide bonds. Egg allergy is the second most common food allergy with a prevalence of up to 1.7 % of children and adults on the basis of the available studies involving double-blind placebo-controlled food challenges . ClinicaEgg white is the main source of allergens in egg. Four of them, named from Gal d 1 to Gal d 4, have been identified as the major ones, with ovomucoid (OM) , which makes up to 11% of the egg white, being considered the immunodominant as judged by its binding frequency to IgE from allergic patients. OM from hen egg is a glycoprotein with trypsin inhibitor activity, a molecular mass of approximately 28.0 kDa, and an isoelectric point of 4.1. Its polypeptide chain consists of 186 amino acids, forming three structurally independent tandem domains each of 60 amino acids in length . One particular characteristic of OM is its high carbohydrate content, which is between 20–25%, with two carbohydrate chains on each of the first and second domains and one chain present on about 50% of the third domain . Thus, aIn the case of many food allergies, particularly to plant proteins, antibodies specific to carbohydrate determinants are frequently found, although they appear not to have clinical relevance . In any The aim of the present work was to assess the involvement of the carbohydrate moieties of OM in its allergenic properties and digestibility. To that end, OM was enzymatically deglycosilated and, using blood from egg-allergic patients, the IgE-binding and basophil activation properties of the glycosylated and deglycosylated forms were compared. Glycosylated and deglycosylated OM were hydrolysed with a physiologically relevant model by mimicking three areas of the gastrointestinal tract: the mouth, stomach and small intestine, the nonglycosylated peptides obtained were identified by RP-HPLC-MS/MS and the IgE-binding properties of the most relevant resulting fragments were evaluated.All human samples were obtained with written consent from the donors (in case of adults) or from the next of kin, caretakers, or guardians on the behalf of the minors/children involved in the study. The Bioethics Committee from the Consejo Superior de Investigaciones Científicas (CSIC) approved all experiments.2, 0.04% NaCl, pH 7.5, at a concentration of 20.7 mg/mL and deglycosylated with PNGase F  at 37 °C, with constant stirring for 24h. PNGase F was added again to the solution at the same ratio and incubated for a further 4 days at 37 °C. A control without PNGase F was also included.OM from chicken egg white  was dissolved in 5 mM potassium phosphate, 4 mM CaClFree sugars were removed from deglycosylated OM (dOM) by centrifugation at 4000g for 5 min at 4 °C in ultrafiltration devices of 10.000 Da cut off , until no absorbance was detected at 490 nm in the permeates . ProteinSDS-PAGE of OM, dOM and their digests was performed on Precast Criterion 4-12% and 12% Bis-Tris gels  and electrophoretic separations were carried out at 150 V, using XT-MES as running buffer (Bio-Rad), Samples were diluted to 1 and 5 mg/mL in sample buffer containing 2% (w/v) SDS and 5% (v/v) β-mercaptoethanol and heated at 95°C for 4 min. Gels were stained with Bio-Safe Coomassie G-250 (Bio-Rad) or Periodic Acid Schiff (PAS).Circular dichroism (CD) spectra were obtained in a Jasco J-810 spectropolarimeter . Far (195-260 nm) and near (250-350 nm) UV CD spectra of OM and dOM, in phosphate buffer 50 mM pH 7.0, were recorded at 20 °C using cells with respective path lengths of 0.1 and 0.2 cm. Spectra represent the average of three accumulations collected at 20 nm/min, with a 2 s time constant, a 0.2 nm resolution, and a sensitivity of 100 mdeg. The samples were dissolved at 0.2 mg/mL for the analysis in the far-UV region and at 0.5 mg/mL for the near-UV region. The buffer blanks were subtracted from each CD spectrum. Empirical determinations of protein secondary structure were obtained employing the CDNN secondary structure analysis software . Human IgE-binding of OM, dOM and their digests was assessed by inhibition ELISA (using OM as coating antigen) as previously reported . IndividWestern Blotting following SDS-PAGE of OM and dOM and their hydrolysates was performed as described , using tTM Lysing Solution (BD Biosciences), and acquired in a Gallios Flow Cytometer (Beckman Coulter). Anti IgE antibody  and fMLP (Sigma-Aldrich) were used as positive control. Percentage of activation upon stimulation with OM and dOM was calculated and normalized according to the value obtained for RPMI-IL3 (negative control). Basophil activation assays were performed as described by Martos et al , with soin vitro oral digestion, OM and dOM were dissolved in simulated saliva fluid  at a concentration of 18.8 mg/mL. After incubation at 37 °C for 15 min, α-amilase  was added at the physiological ratio of 150 U/mL of simulated fluid. Oral digestions were performed at 37 °C during 2 min and stopped by decreasing the pH to 3.5 with 1N HCl.For In vitro gastric and duodenal digestions were performed according to [3.rding to  with somrding to . Gastric2 and 0.250 M bile salt mixture. After preheating at 37 °C for 15 min, pancreatic porcine lipase , pancreatic porcine colipase (EC 259-490-1), pancreatic bovine trypsin , and pancreatic bovine α-chymotrypsin   were added to the duodenal mix [2 and 20.3 mM Bis-tris. Duodenal digestions were performed on the 60 min gastric digests re-adjusted to pH 6.5, with the addition of 0.25 M Bis-tris, pH 6.5, 1 M CaClenal mix . The reaTM . The m/z spectral data were processed and transformed to spectra representing mass values. BioTools  was used to process the MS(n) spectra and perform peptide sequencing.RP-HPLC-MS/MS analyses of the digested samples, after a reducing step using 70 mM dithiothreitol (DTT) at pH 7.0 for 1 h at 37 °C, were performed on an Agilent 1100 HPLC System  with a RP318 C18 column . The HPLC system was connected on-line to an Esquire 3000 quadrupole ion trap  equipped with an electrospray ionisation source. Operating conditions were as follows: solvent A, 0.37 mL/L TFA in Milli-Q water and solvent B, 0.27 mL/L TFA in HPLC grade acetonitrile; flow rate, 0.8 mL/min; injection volume, 50 μL. A linear gradient of solvent B in A, from 0 to 60% in 60 min, followed by 60% B for 30 min was used. Mass spectra were recorded over the range 100–3000 m/z using Data Analysis17 peptides selected from in vitro digestions were commercially synthesized . Among the deglycosylated peptides identified in the gastroduodenal digests of OM and dOM, the peptides shorter than 8 amino acids, those which were part of longer sequences and/or the peptides that overlapped with well-established epitopes of OM by microarray and SPOT membrane in previous studies, were discarded. Peptides with 8 and 9 amino acids were extended to 10 amino acids with the preceding and following amino acids of the protein chain and the peptides over 20 amino acids were replaced by 2 shorter overlapping peptides. The nitrocelullose membranes were conditioned in Tris buffer  for 20 min, and 1µl from each peptide was spotted onto the membrane and allowed to dry. Then, the nitrocellulose membranes were blocked with Tris Buffer Saline containing 0.05% v/v Tween 20  with 1% w/v BSA, for 60 min and washed with TBST. Immunolabelling with ten individual serum samples . Student t-test was used to compare between groups. Data were considered statistically significant for P values less than 0.05.To assess the effectiveness of deglycosylation SDS-PAGE with Coomassie G-250 and PAS staining was conducted. As shown in  We further checked by CD-spectroscopy that, in addition to an efficient removal of N-linked oligosaccharides, PNGase F treatment maintained the degycosylated protein in its native structure. The far-UV CD spectrum of OM was similar to that reported by  and did IgE-binding to OM and dOM was evaluated by inhibition ELISA using the sera from 10 egg-allergic patients 1-10 in . As depiIn order to investigate the IgE specificity, sera from the same 10 patients were used to perform Western blot analyses . IgE froThe capacity to trigger basophil activation was determined on PBMCs from 6 egg-tolerant adult donors passively sensitized with a pool of sera from patients 1, 3, 5, 7 and 9 . The resin vitro oral, gastric and duodenal digestions. After the 2 min-oral phase, the SDS-PAGE patterns from both OM and dOM showed a much broader shape, which could be attributed to an incomplete reduction of the nine disulfide bridges caused by the low sample pH (pH 3.5 was used to stop α-amilase action), which diminished their mobility in the acrylamide gel [mide gel . HoweverAs shown in The IgE-binding of the digests was evaluated by inhibition ELISA using the sera from patients 11-16 . The imm10 and Asn75 were detected in the OM hydrolysates  probably due to a partial deglycosylation of the commercial sample used. The peptide patterns of the hydrolysates produced were very similar, showing 56 peptides in common after the simulated orogastrointestinal digestion. This indicated that, while deglycosylation enhanced susceptibility to proteolysis, no major differences in the cleavage sites of the protein were detected after hydrolysis times representative of the transit times in the stomach and duodenum. In the N-terminal region, around the glycosylated Asn10, pepsin hydrolysed OM after Ala12 and dOM after Phe8, Asp13 and Glu15. Very few peptides were identified in the region between the glycosilated positions Asn53 and Asn75 even in the deglycosylated form  and duodenal (30 min) digests of OM and dOM and ted form . In this Considering the similarities between the peptide patterns of the orogastroduodenal digests of OM and dOM, 17 representative peptides, common to both hydrolysates, were selected as explained in the method section and chemically synthetized. Their IgE binding is shown in N-linked glycoproteins, without secondary or tertiary structural changes as determined by CD spectroscopy (OM was successfully deglycosilated using microbial PNGase F , an amidtroscopy . PreviouIn the present study, 80% of the patients showed lower IgE binding to dOM as compared with OM . The maiIn accordance with the lower IgE binding, we found a subtle reduction in the percentage of activated basophils after incubation with dOM as compared to OM . There ain vitro oral digestion, but completely degraded by gastric digestion ( The simulated digestion experiments showed that OM and dOM were not affected by igestion . A profi20-Val21 and Ala133-Val134 were cleaved by pepsin in our system (Kovacs-Nolan et al.  describer system  and thusr system  The abser system . Takagi  Little to no change in the band pattern was observed during the duodenal phase of digestion, likely because the peptides released by pepsin action retain trypsin inhibitory activity that helps to maintain OM peptide fragment integrity . Followi A further investigation on whether the peptides resulting from orogastrointestinal digestion of OM contained IgE-binding epitopes revealed that two regions of the protein, that is those within the residues 80-89 and 133-180, were recognized by more than 70% of the allergic patients studied, while from 40 to 70% of the patients recognized two other regions, 36-61 and 100-122 . Most ofin vitro digests of OM and dOM was low, the evaluation of the presence of potential epitopes among the nonglycosylated orogastroduodenal products of digestion of OM and dOM revealed the presence of high-frequency IgE-binding fragments that could remain linked by disulphide bonds.In conclusion, this work provides evidence for an enhanced IgE reactivity towards carbohydrate containing OM in some egg-allergic patients that can be due to cross-sensitization, but also to sensitization to the glycosylated components. In addition to a direct implication of the carbohydrate chains of OM on its IgE binding, whose clinical relevance remains to be established, they contribute to an increased resistance to proteolysis, particularly during the first stages of gastric digestion, which may play a role in its allergenic potency. Although the residual IgE binding of the