The fibroblast growth factor (FGF) system plays a critical role in the maintenance of vascular integrity via enhancing the stability of VE-cadherin at adherens junctions. However, the precise molecular mechanism is not well understood. In the present study, we aimed to investigate the detailed mechanism of FGF regulation of VE-cadherin function that leads to endothelial junction stabilization.In vitro studies demonstrated that the loss of FGF signaling disrupts the VE-cadherin-catenin complex at adherens junctions by increasing tyrosine phosphorylation levels of VE-cadherin. Among protein tyrosine phosphatases (PTPs) known to be involved in the maintenance of the VE-cadherin complex, suppression of FGF signaling reduces SHP2 expression levels and SHP2/VE-cadherin interaction due to accelerated SHP2 protein degradation. Increased endothelial permeability caused by FGF signaling inhibition was rescued by SHP2 overexpression, indicating the critical role of SHP2 in the maintenance of endothelial junction integrity.These results identify FGF-dependent maintenance of SHP2 as an important new mechanism controlling the extent of VE-cadherin tyrosine phosphorylation, thereby regulating its presence in adherens junctions and endothelial permeability. Regulation of endothelial permeability is crucial for many essential vascular functions including the passage of molecules and cells through the endothelium without altering structural integrity of blood vessels The maintenance of the vascular barrier function is largely achieved by endothelial cell junctions which are comprised of a complex network of adhesive proteins organized into tight junctions and adherens junctions We have recently found, using in vitro and in vivo approaches, that inhibition of fibroblast growth factor (FGF) signaling impairs vascular integrity in the adult vasculature Antibodies against the following antigens were commercially obtained: VE-cadherin, p120-catenin, SHP2, PTPβ, DEP-1, PTP1B, c-Src (Santa Cruz Biotechnology), PTPµ, phospho-tyrosine 416 Src, phospho-tyrosine 527 Src, Fak, phospho tyrosine 397 Fak , phospho-tyrosine 658 VE-cadherin, phospho-tyrosine 731 VE-cadherin (invitrogen), HA (COVANCE), and β-tubulin (Sigma). Anti phospho-tyrosine 685 VE-cadherin antibody was generated in our laboratory.2 in EGM-2 medium (Lonza). Adenovirus carrying the truncated form of FGF receptor 1 lacking the cytoplasmic part of the receptor (FGFR1DN) was generated as previously described Bovine aortic endothelial cells (BAEC) and Human umbilical vein endothelial cells (HUVEC) were purchased from Lonza, and were cultured at 37°C in 5% COcDNA of human VE-cadherin fused in-frame with GFP at the C-terminus (VE-cadherin-GFP) was a kind gift from Dr. Sunil K. Shaw  Human SHP2 constructs (wild-type and C/S mutant) were purchased from Addgene and subcloned into pLVX-IRES-puro lentiviral expression vector (Clontech). Lentivirus packaging and envelope vectors, pMDLg/pRRE, pRSV-Rev and pMD2.G, were also purchased from Addgene. These plasmids were cotransfected into HEK-293T cells to produce a recombinant lentivirus stock 2. To obtain stable infected cells, the cells were selected with 1 µg/ml puromycin.BAEC on a 6-well plate were cultured in EGM-2 medium (Lonza) to 50–60% confluent. Lentivirus was added to the cells with 5 µg/ml polybrene and the 6-well plate was centrifuged at 2300 rpm for 90 min at 37°C following an 8-hour incubation at 37°C with 5% COImmunocytochemistry was performed with a standard procedure using antibodies described above. Cells were fixed in 2% paraformaldehyde in phosphate buffered saline (PBS) for 15 min at room temperature (RT), permeabilized in 0.1% Triton-X in PBS for 5 min at RT, and blocked in Image-iT FX signal enhancer (Invitrogen) for 30 min at RT. Coverslips were incubated in primary antibody at 4°C overnight, washed three times in PBS and then incubated with Alexa Fluor-conjugated secondary antibodies (Invitrogen) for 1 hour at RT. Then the coverslips were washed three times in PBS and mounted using Fluoro-Gel (Electron Microscopy Sciences). Images were captured using Zeiss 510 laser scanning confocal microscopy. Gap formation of the monolayer was analyzed using immunostaining images in which the proportion of the gap area between cells to the total image area was calculated using NIH Image J software.2) and connected to its recorder device. Monolayer resistance was recorded at 15 kHz in 5-minute intervals.Transendothelial electrical resistance, an index of endothelial cell barrier function, was measured in real time using an electric cell-substrate impedance sensing (ECIS) system  Cells were washed twice with ice-cold PBS and lysed in RIPA buffer: 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% NP40, 0.5% sodium deoxycholate, 0.1% SDS, and 1× Complete protease inhibitor mixture (Roche) and PhosSTOP Phosphatase inhibitor cocktail (Roche). For evaluation of VE-cadherin phosphorylation, lysis buffer was supplemented with freshly prepared pervanadate (300 µM) 5′-TGATTCGCTGTCAGGAACTG; reverse 5′-TGTGCCCAATGTTTCTACCA-3′, bovine GAPDH, forward 5′-GGCGCCAAGAGGGTCAT-3′; reverse 5′-GTGGTTCACGCCCATCACA-3′, using Ssofast EvaGreen Supermix (Bio-Rad) on a CFX96™ Real-Time PCR Detection System.RNA preparation and cDNA synthesis were described previously t test. Results were considered significant at P<0.05.Data were expressed as mean ± SD. Comparison between groups were performed with a two-tailed Student’s To investigate the role of FGF signaling in the vasculature, we employed a dominant negative construct of FGF receptor 1 (FGFR1DN) capable of heterodimerizing with all four isoforms of FGF receptors, thereby inhibiting overall FGF signaling in vitro and are thought to be involved in the regulation of endothelial permeability and leukocyte transmigration Previous reports support the idea that VE-cadherin stability at adherens junctions is dependent on the phosphorylation status of VE-cadherin, which modulates VE-cadherin/p120-catenin interaction required for the retention of the VE-cadherin complex at cell-cell contacts To further confirm the FGF effect on VE-cadherin phosphorylation, we employed a soluble FGF receptor (FGF-trap) as an alternative means to inhibit FGF signaling. sFGFR has been extensively evaluated and shown to be an effective inhibitor of FGF signaling both in vitro and in vivo Next we investigated the molecular mechanism of FGF-dependent regulation of VE-cadherin phosphorylation. Src has been identified as a kinase responsible for VE-cadherin Y658 phosphorylation Since activity of two known kinase controlling VE-cadherin phosphorylation was not affected, we then evaluated the possibility that a PTP, but not a kinase, is responsible for modulating Y658 VE-cadherin phosphorylation. Among PTPs, DEP-1 (CD148), VE-PTP (PTPβ), PTPµ, PTP1B and SHP2 have been implicated in the interaction with the VE-cadherin-catenin complex and/or regulation of VE-cadherin function by modifying its phosphorylation status These results suggest the possibility that SHP2 bound to VE-cadherin may regulate the phosphorylation level of VE-cadherin Y658 site. To formally evaluate this, we tested the effect of inhibition of SHP2 activity on VE-cadherin Y658 phosphorylation. Expression of the C/S mutant of SHP2  in BAEC led to an increase in VE-cadherin Y658 phosphorylation compared to SHP2 overexpressing cells or control cells , while oTo determine the mechanism of SHP2 down-regulation by FGF signaling shutdown, we next tested whether FGF signaling controls SHP2 expression at the transcriptional or post translational level. Quantitative analyses of SHP2 mRNA levels using real-time quantitative PCR in BAEC transduced with Ad-FGFR1DN showed an increase in mRNA abundance while the control virus had no effect . This exSince our observation indicates that SHP2 expression is impaired in cells transduced with Ad-FGFR1DN, we tested whether SHP2 overexpression can rescue the phenotypes of endothelial cells lacking FGF signaling. Adenoviral transduction of SHP2 into Ad-FGFR1DN-tranduced BAEC resulted in a reduction of Y658 VE-cadherin phosphorylation  and restThe results of this study demonstrate that increased permeability of the endothelial monolayer after suppression of FGF signaling is due to the loss of adherens junctions mediated by a decrease in SHP2 expression that directly leads to increased VE-cadherin phosphorylation at the Y658 site and the loss of VE-cadherin-p-120 binding. Several lines of evidence support this conclusion. Most directly, adenovirus-mediated SHP2 overexpression can rescue increased permeability, increased Y658 phosphorylation and the loss of VE-cadherin-p120 binding caused by inhibition of FGF signaling. FGF signaling controls SHP2 protein levels by inhibiting lysosome-mediated SHP2 degradation with the absence of FGF signaling input leading to a markedly shortened SHP2 half-life. Finally, the key role of Y658 site phosphorylation is demonstrated by the rescue of the Ad-FGFR1DN phenotype by Y658F VE-cadherin mutant. Taken together, these data demonstrate that FGF signaling potentiates VE-cadherin stability at endothelial junctions by regulating SHP2 ability to restrain tyrosine phosphorylation of VE-cadherin.Many previous studies support the idea that tyrosine phosphorylation of VE-cadherin and other components of adherens junctions is associated with impaired barrier function. Permeability-increasing agents such as histamine, platelet-activating factor and VEGF all induce tyrosine phosphorylation of VE-cadherin-catenin complex These results suggest a possibility that an alternation in PTP rather than kinase activity is the principal driver of the phenotype. Unlike kinases, PTP activity is largely determined by the expression level or localization and not by a specific phosphorylation event. A number of PTPs including DEP-1, VE-PTP (PTPβ), PTPµ, PTP1B and SHP2, have been reported to directly or indirectly associate with VE-cadherin and/or adherens junction components. PTPµ is reported to localize almost exclusively at endothelial junctions where it associates directly with VE-cadherin in both human pulmonary artery and lung endothelial cells SHP2 has also been shown to associate with VE-cadherin via β-catenin How SHP2 is recruited to VE-cadherin-catenin complex and which tyrosine phosphorylation of VE-cadherin is regulated by SHP2 is of special interest. One study shows that SHP2 is recruited to VE-cadherin when VEGF receptor tyrosine kinase phosphorylates VE-cadherin. The VE-cadherin-bound SHP2 induces release of Csk from VE-cadherin potentially by dephosphorylating its VE-cadherin binding site (Y685). Consequently, c-Src Y527 phosphorylation level decreases, and c-Src Y416 phosphorylation level increases which in turn results in c-Src activation While we show that FGF signaling is required for VE-cadherin-SHP2 interaction, certain aspects of this regulation remain uncertain. We found that downregulation of SHP2 expression in the absence of FGF signaling is due to shortened protein half-life and that inhibition of the lysosomal pathway, but not the proteosomal pathway, restored the SHP2 expression levels of cells lacking FGF signaling. Although we also examined the contribution of autophagy in this process, we were not able to obtain the evidence suggesting that FGF signaling controls autophagy in endothelial cells. Therefore, we concluded that SHP2 is degraded via lysosomal pathway in the absence of FGF signaling; however, how FGF controls this process is unclear. Furthermore, the precise mechanism responsible for FGF-dependent association with SHP2 with VE-cadherin is uncertain. One possibility is that the latter can occur via the VEGF signaling pathway since VEGF can recruit SHP2 to VE-cadherin-catenin complex Finally, it is unclear which FGF is responsible for continuous stimulation of the endothelium necessary for the maintenance of SHP2 expression. Endothelial cells express all four FGF tyrosine kinase receptors and numerous FGF ligands are present in the serum in vitro and in blood and interstitial fluid in vivo. Given significant redundancy in the FGF signaling pathways where multiple ligands can activate multiple receptors, it is virtually impossible to identify a specific FGF that can be responsible for this effect. In fact it is likely that multiple redundant ligands are capable of performing this vital function.In summary, we report a novel function of FGF signaling in the vasculature that involves FGF-dependent maintenance of endothelial SHP2 levels and plays a key role in the regulation of endothelial barrier function.


Using the SREBP-1a expressing human hepatoma cell line HepG2 we have shown previously that human SREBP-1a is phosphorylated at serine 117 by ERK-mitogen-activated protein kinases (MAPK). Using a combination of cell biology and protein chemistry approach we show that SREBP-1a is also target of other MAPK-families, i.e. c-JUN N-terminal protein kinases (JNK) or p38 stress activated MAP kinases. Serine 117 is also the major phosphorylation site in SREBP-1a for JNK. In contrast to that the major phosphorylation sites of p38 MAPK family are serine 63 and threonine 426. Functional analyses reveal that phosphorylation of SREBP-1a does not alter protein/DNA interaction. The identified phosphorylation sites are specific for both kinase families also in cellular context. To provide direct evidence that phosphorylation of SREBP-1a is a regulatory principle of biological and clinical relevance, we generated transgenic mice expressing mature transcriptionally active N-terminal domain of human SREBP–1a variant lacking all identified phosphorylaton sites designed as alb-SREBP-1aΔP and wild type SREBP-1a designed as alb-SREBP-1a liver specific under control of the albumin promoter and a liver specific enhancer. In contrast to alb-SREBP–1a mice the phosphorylation–deficient mice develop no enlarged fatty livers under normocaloric conditions. Phenotypical examination reveales a massive accumulation of adipose tissue in alb-SREBP-1a but not in the phosphorylation deficient alb-SREBP-1aΔP mice. Moreover, preventing phosphorylation of SREBP-1a protects mice also from dyslipidemia. In conclusion, phosphorylation of SREBP-1a by ERK, JNK and p38 MAPK-families resembles a biological principle and plays a significant role,  Sterol regulatory element binding proteins (SREBPs) are the predominant transcription factors controlling the synthesis of cholesterol and fatty acids in liver Furthermore the SREBP-1 isoforms differ in the expresson levels, e.g. transcripts of SREBP-1c are approximately 10 fold higher abundant in liver as SREBP-1a One essential feature of SREBPs is that they are transcribed and translated into inactive precursor molecules which are embedded in the membrane of the cellular endoplasmatic reticulum. The composition of this pool still remains unclear and an accumulation of either SREBP isoform, even if the expression rates are reduced cannot be excluded. To generate the transcriptional active domain, SREBPs are released by a complex two-step sequential proteolytic machinery initiated by cholesterol depletion of the cell. Only the mature forms of SREBPs translocate in the nucleus and activate target genes It is well known that other MAPK cascades, like JNK  as well as p38 MAP kinases, target transcription factors directly by phosphorylation, thereby altering their transactivity, leading to changes of cellular gene regulatory networks Since SREBP-1a is the master regulator of both pathways, i.e. synthesis of fatty acids as well as cholesterol, we had the hypothesis, that SREBP-1a is phosphorylated by JNK and p38 MAPK thereby altering transcriptional activity and cellular lipid metabolism. Accordingly in this report we show, that SREBP–1a is directly phosphorylated by members of stress kinases families, i.e JNK and p38 MAPK. Using protein chemistry methodology we identify the major kinase specific phosphorylation. In HepG2 cells we have shown, that identified phosphorylation sites are also targets in cellular context. Furthermore, to study physiological and clinical impact of SREBP-1a phosphorylation, we generated mouse models that overexpress the transcriptional active wild type domain of SREBP-1a or a construct in which all identifed MAPK phosphorylation were destroyed by mutation in liver. Using these transgenic mouse models we provide first evidence that phosphorylation of SREBP–1a at these site appears to be of physiological relevance.Previously we have shown that the N-terminal domain of human SREBP-1a is a substrate for ERK kinase and identified S117 as the major phosphorylation site The profile of SREBP-1a-NT phosphorylated by p38 was completely different and more complex . Using tin vitro phosphorylated radiolabelled GST-SREBP-1a-NT by reversed phase mass spectrometry. The radiolabelled peak 1 phosphopeptide detected in an UV elution profile . The mice were bred on C57Bl6 background which also served as global controls throughout the study. Viability, fertility or breeding efficiency and nesting behaviour of the transgenic animals was not affected and the observed life span was not reduced.To investigate the biological relevance of MAPK related phosphorylation of human SREBP 1a EBP-1aΔP . In addiThe presence of the transgenes was monitored by genotyping of the intersection of albumin promoter and SREBP-1a coding sequence . As mousTo determine the functionality of the liverspecific overexpression of the human transcriptional active domain of SREBP-1a and the impact of SREBP-1aΔP the gene expression of key metabolic enzymes was analyzed in liver of these animals  as a funth week  in liver of alb-SREBP-1a mice was approximately fourfold higher as in liver of C57Bl6 mice. In contrast the phosphorylation deficient mice were mostly protected against lipid accumulation and show a 1.5 fold- increase. Detailed analyses of fatty acid composition in liver tissues and serum were performed . In liveThe mice overexpressing functional SREBP-1a in liver developed a fatty liver, but the phenotype is abrogated if the main phosphorylation sites in SREBP-1a were mutated as in alb-SREBP-1aΔP mice. Excess intracellular lipid accumulation in hepatocytes impairs liver functionality and insulin sensitivity. Caculating of HOMA-IR index as surogate for insulin resistance indicated that alb-SREBP 1aΔP mice have a comparable level to C57Bl6, but the variation within this genotype is broarder. The HOMA-IR index of the alb-SREBP 1a mice indicated the beginning of insulin resistance. QUICKI as surogate for insulin sensitivity indicated for alb-SREBP 1aΔP that the insulin sensitivity was in normal range but with larger varaition that the controls. In contrast for alb-SREBP 1a mice the insulin sensitivity and ß-cell failure were significantly pronounced . So alsoWether obesity or ectopic hepatic lipid accumulation are associated to inflammation is still under debate No genotype specific cyokine pattern can be detected in serum of alb-SREBP-1a and alb-SREBP-1aΔP mice, nevertheless the abundance of some cytokines is altered. Again the alb-SREBP-1aΔP show a cytokine profile more comparable but not totally identical to C57Bl6. An increase in CSF-2 and CXCL-1 and a reduction in TREM-1 can be otained. In contrast, significant alterations in the alb-SREBP-1a mice is the reduced abundance of the respective chemokines exept for C5a and sICAM where no alteration can be obtained.SREBP-1a has been identified as a connective link at the gene regulatory level integrating signaling of various hormones, cytokines, and metabolites Here, we provide direct evidence that SREBP–1a is a selectively phosphorylated target of JNK and p38 signaling cascades and might link MAPK cascades to lipid metabolism.MAPK cascades are the paradigm of activiation by sequential phosphorylation with high target substrate specificity. Previously, we have shown that the SREBP transcription factors are targets for intracellular signaling pathways Phosphorylation of SREBP-1a might also affect other posttranslational modifications. The work of Sundqvist et al. In general the N-terminal transcriptional active domain of SREBP-1a has a basal transcriptional activity. In cell culture experiments increasing the amount of SREBP-1a by overexpression is sufficient to induce transcription. This basal transcriptional activation can be synergstically increased if there is a further induction i.e. a hormonal stimulus to initiate the signalling cascades to phosphorylation The protein pool in the nuclear envelop seems to provide sufficient mature SREBP–1a for posttranslational modification. Previously we have shown that phosphorylation of SREBP-1a does not interfere with mRNA expression, maturation or protein/DNA interaction of SREBP-1a, but is related to ERK-MAPK pathway inducible increase of e.g. gene expression of SREBP-1a target genes like the LDLR and central genes of lipid metabolism in cell culture models in vivo we have created mouse models which express specifically in liver either the mature form of SREBP–1aΔP, which is deficient in all identified MAP kinase phosphorylation sites, or the wild type SREBP–1a. Due to the complex cholesterol dependant feedback regulation of SREBP–1a secretion from membranes and the fact that cellular cholesterol overload restrains the SCAP–SREBP–1 complexes to the membranes, we overexpressed the transcriptional active N–terminal domain directly, circumventing secondary effects due to the complex and highly regulated releasing cascade of mature SREBP–1a The limitation of cell culture models always is that they can't answer the question whether a mechanism identified has biological importance and possibly allows the explanation of a pathophysiological process. To adress the potential biological importance of SREBP-1a phosphorylation Clinical studies have shown that the intracellular amount of lipid in liver is associated with insulin resistance. Therefore syndromes like the non alcoholic fatty liver disease appears to be a hepatic manifestation of insulin resistance or the metabolic syndrome The pattern of circulating cytokines are a indicator for the physiological condition of the liver. There is ongoing discussion of the role of inflammation in the pathogenesis of obesity and fatty Taken together, we have shown that SREBP-1a is phosphorylated by JNK and p38 MAPK at different specific sites. Furthermore, direct evidence is provided that phosphorylation does not influence DNA binding but phosphorylation of the identified sites plays a role in stress mediated effects on LDLR promoter activity or activation and therefore affects target gene expression. These data further support our concept that, besides sterol-dependent cleavage, SREBPs are regulated by phosphorylation affecting their trans-activity by various MAPK cascades leading to changes of cellular gene regulatory networks Our investigations implicate that in liver phosphorylation of SREBP-1a has biological implications on the development of fatty liver and massive weight gain. This is impressively demonstrated by the phosphorylation deficient SREBP-1aΔP animals which are are protected against all facets of the phenotype including lipid accumulation, visceral adipositas and signs of persisting insulin reristance. Phosphorylation of SREBP-1 also has clinical implications as we have recently reported a rare heterozygous mutation in a patient with severe dyslipidemia hampering specifically phosphorylation of Erk- and JNK-MAPK specific S117 in SREBP-1a N-terminal domain of human sterol regulatory element binding protein (SREBP)-1a was inserted in frame to glutathione-S-transferase (GST) into pGEX 3X to generate SREBP-1a-NT GST-fusion protein as previously described 2). The reaction was initiated by addition of 50 µM [γ32P] ATP (10 Ci/mmol) in a final volume of 40 µl kinase buffer. The reaction was terminated after 15 min at 30°C by addition of 5× SDS loading buffer. The phosphorylated proteins were separated by 10% SDS-PAGE and analyzed by autoradiography of the coomassie-stained dried gels.GST-SREBP-1a-NT or mutated GST-SREBP-1a-NT  fusion proteins were by expressed in E. coli strain BL21-codon plus (DE3)-RP (Stratagene) and purified according to the manufacturer's recommendations . Phosphorylation of 10 µg GST-SREBP-1a-NT fusion protein or 0.5 µg PHAS by MAPK JNK1, JNK2, p38α, p38β and p38γ  were performed using activated GST-JNK1, -JNK2, -p38α, -p38β or -p38γ fusion protein (40 ng/µg substrate) in kinase buffer . The His-tagged SREBP-1a-NT was purified using Ni-NTA magnetic agarose beads (Qiagen). For phosphorylation 200 ng of isolated protein was incubated with recombinant activated JNK1 or p38α (40 ng/µg substrate) in kinase buffer according to supplier's instruction manual (Upstate Biotechnology). To control for phosphorylation efficiency, kinase reaction was performed with an aliquot of His-SREBP-1a-NT using additionally 10 µCi [γ32P] ATP.For electro mobility shift assay 1 µg pcDNA3.1/HisA-SREBP-1a-NT was 4CH2COOH pH 7.0) and 0% of buffer B (0.5 M KH2PO4 pH 4.0). Ratios of B were increased to 10% in 40 min and from 10% to 50% in 75 min. Fractions of 0.5 ml were collected. Cerenkov counting in scintillations counter (Beckman Coulter) determined the relative amount of radiolabeled peptides.10 µg of GST-SREBP-1a-NT or mutated GST-SREBP-1a-NT fusion proteins phosphorylated by JNK1, JNK2, p38α, p38β or p38γ as described above were separated on a 10% SDS-PAGE and phosphorylated proteins were in gel digested by trypsin (Promega) over night. The peptides were eluted with 50 mM ammoniumcarbonate and separated on an anion exchange column  using Beckman gold solvent delivery system . The HPLC flow rate was 0.5 ml/min. After injection of 1 ml sample the peptides were eluted beginning at 100% buffer A (20 mM NH18-reversed phase column. Fractions containing radiolabeled peptides were subjected to Edman degradation and MALDI-TOF (time of flight) mass spectrometry. Positive ion mass spectra were acquired in a linear and reflector mode using an acceleration voltage of 20,000 V. In addition to manual interpretation of post source decay (PSD) fragment ion spectra the SEQUEST™ algorithm was used to identify the peptide phosphorylation sites. For database search the OWL sequence database (University of Leeds) was used.Reversed phase and MALDI-MS analyzes of GST-SREBP-1a-NT fusion proteins phosphorylated by p38α (40 ng/µg substrate) with 250 µM ATP  as described above were performed essentially as described Protein/DNA binding reaction was performed as described with radiolabelled double-stranded oligonucleotide containing the LDLR promoter consensus sre-1 fragment and ADD1/SREBP-1c consensus E-box fragment 5 cells/well) was mixed with reporter plasmid pG5-luc (0.5 µg/well), with pFA/Gal4-SREBP-1a-NT, pFA/Gal4-SREBP-1a-NT S117A, pFA/Gal4-SREBP-1a-NT S63A/T426V, pFA/Gal4-SREBP-1a-NT S63A/S117A/T426V or pFA/CMV vector (25 ng/well), with pFC/MEK3, pTREx/MKK4 or pFC vector (25 ng/well) and pRL(-mcs) (0.1 µg/well) for control of transfection efficiency, respectively. Then plasmids were transferred to an electroporation cuvette and electroporated. Before seeding on six-well plates cell suspension was diluted with standard medium. On day 1 after transfection cells were cultured in lipid deficient medium (RPMI-1640) containing, 0.5% lipoprotein deficient serum (LPDS) ) for 16 h and treated as indicated below. Cell lyses and measurements of firefly as well as renilla luciferase activities of cell extracts (20 µl/probe) were performed according to supplier's instructions (Promega).HepG2 cells  were maintained in standard medium  fetal calf serum (FCS) and antibiotics (Invitrogen). Promoter reporter gene analyses were performed as described 2, 0.5 mM DTT, 1 mM EDTA, 1 mM EGTA) supplemented with protease inhibitor cocktail  and phosphatase inhibitor cocktail . Samples were lysed by shearing 10 times through a 25-gauge needle and centrifuged . The resulting nuclear pellet was resuspended in hypertonic buffer ) supplemented with protease inhibitor cocktail and phosphatase inhibitor cocktail and incubated on a thermomixer . The extracts were centrifuged  and aliquots of supernatant, used as nuclear extract, were frozen in liquid nitrogen and stored at −80°C. Protein extracts were resolved on 7.5% SDS-PAGE and electroblotted to nitrocellulose membrane  blocked and probed with mouse monoclonal antibody against HA peptide (YPYDVPDYA) conjugated with peroxidase . A polyclonal SREBP-1 antibody  was used to determine SREBP-1 precursor protein and the polyclonal SREBP-1 antibody  was used to determine the complete nuclear SREBP-1 contends. Visualization was performed with ECL™ plus western blotting detection reagents according to manufacturer's instructions  and a Versa Doc instrument .To detect precursor and nuclear SREBP-1 in liver and HA-SREBP-1aΔP or HA-SREBP-1a in liver and other tissues of the transgenic animals at protein level, 10 mg of snap frozen tissue samples  were pottered (10 sec) in 250 µl hypotonic lysis buffer  and SREBP-1a-NT (5′-TAGGCCAGGGAACTGACTG-3′) specific primers. The presence of the constructs was determined with a DNA 1000 kit on 2100 Bioanalyzer according to manufactures instructions .To generate transgenic animals approximately 450 fertilized C57Bl6 oocytes were microinjected with alb-HA-SREBP-1aΔP or alb-HA-SREBP-1a construct and placed into eight foster mice. Of the offspring 5 founders were tested positive for the HA-SREBP-1aΔP and the HA-SREBP-1a construct, respectively. To screen the presence of transgene ear biopsies were taken of the animals at weaning and genomic DNA was extracted with a tissue kit (Qiagen) according to manufactures instructions. Routinely 50 ng DNA was used for PCR with albumin promoter  and they had free access to water. Weekly the animals were controlled for weight gain; food or water intake was also monitored. At the age of 24 weeks alb-SREBP-1aΔP as well as alb-SREBP-1a mice and age matched C57Bl6 mice were sacrificed by CO2 asphyxiation. Mice of each genotype of the German Diabetes Center colonies were also transferred to the collaborating Institute for Diabetes Research to verify viability, reproducibility and physiological as well as histological parameters in an independent habitat. The Animal Care Committee of the Universities of Duesseldorf and Hamburg approved animal care and procedure .C57Bl6, alb-SREBP-1aΔP and alb-SREBP-1a mice were bred and maintained in our animal facility in a regular 12 h light/dark cycle under constant temperature and humidity . For environmental enrichment the cages were supplemented with nesting material happi-mat®  and a mouse house . At the age of six weeks 12 male littermates of each genotype were kept in colonies of four per standard Macrolon III cage . Mice were fed Blood parameters were measured at 24 weeks of age. Blood glucose was measured with Freestyle™  and triglycerides, cholesterol as well as liver enzymes  were determined on a Hitachie 912 laboratory automat . Serum insulin levels (mU/l) were measured in triplicate by ELISA according to the manufactures recommendation . Fatty acid composition of serum or homogenate of liver biopsies were determined on a Packard model 439 gas-liquid chromatograph equipped with a flame ionization detector and a 60 m Hewlett-Packard fused silica capillary column as described Total RNA of 10 mg freshly isolated mouse liver biopsies was extracted with RNeasy Mini Spin Kit (Qiagen). Real-time (RT)-PCR was performed in triplicate with cDNA equivalent of 20 ng reversed transcribed RNA  per reaction.gene)−Ctgene/(E18S)−Ct18S)*1,000,000 with E = 10−(1/slope).SREBP-1a and SREBP-1c isoform specific RT-PCR was performed with the conserved probe according to Tissues were resected and fixed in 4% paraformaldehyd/PBS for 24 h and embedded in paraffin with automated standard histological procedures . On 3 µm deparaffinized sections standard hematoxylin and eosin (HE) staining was performed. Glycogen storage was monitored with PAS staining . Fibers and extra cellular matrix were visualized using the “van Gierson kit” . For oil red O staining, liver was resected and cryoprotected in 30% sucrose 4°C over night. Thereafter tissue was frozen under liquid nitrogen cooled isopentane and stored in liquid nitrogen until proceeding. For cellular lipid detection, 3 µm sections of tissue were prepared and stained with oil-red-O (Sigma). For characterization of cellular histomorphology, fixed slips were stained in Mayer's hemalaun solution  according to the manufacturer's instructions.For parallel detection of various cytokines, serum of C57Bl6, alb-SREBP-1aΔP and alb-SREBP-1a mice (n = 6 each) were hybridized to array membranes according to the protocol supplied by the manufacturer . Samples of each genotype were analyzed and handeled in parallel troughout the entire procedure. Exposure, data collection and processing of each set of arrays was also performed in parallel . Spots were normalized for internal hybridization positive controls after local background substraction. Normalized data were analysed for genotype specific differences as processed and results per set were used to determine the mean differences in cytokine abundance.Values are presented as means ± SD. Statistical analysis was performed Student T-test or by 2-way ANOVA calculated with Prism 4.03  as indicated.


In addition, PDGF-BB-induced activation of mTORC1, as measured by phosphorylation of the downstream S6 protein, was dependent on phospholipase D (PLD). It has been shown that Erk1/2 MAP-kinase directly phosphorylates and activates mTORC1; in partial agreement with this finding, we found that a Mek1/2 inhibitor delayed S6 phosphorylation in response to PDGF-BB, but it did not block it. Thus, whereas both mTORC1 and mTORC2 are activated in a PI3K-dependent manner, different additional signaling pathways are needed. mTORC1 is activated in a PLD-dependent manner and promotes phosphorylation of the S6 protein, whereas mTORC2, in concert with PLCγ signaling, promotes Akt phosphorylation.Mammalian target of rapamycin (mTOR) can be found in two multi-protein complexes, i.e. mTORC1 (containing Raptor) and mTORC2 (containing Rictor). Here, we investigated the mechanisms by which mTORC1 and mTORC2 are activated and their downstream targets in response to platelet-derived growth factor (PDGF)-BB treatment. Inhibition of phosphatidylinositol 3-kinase (PI3K) inhibited PDGF-BB activation of both mTORC1 and mTORC2. We found that in Rictor-null mouse embryonic fibroblasts, or after prolonged rapamycin treatment of NIH3T3 cells, PDGF-BB was not able to promote phosphorylation of Ser473 in the serine/threonine kinase Akt, whereas Thr308 phosphorylation was less affected, suggesting that Ser473 in Akt is phosphorylated in an mTORC2-dependent manner. This reduction in Akt phosphorylation did not influence the phosphorylation of the S6 protein, a well established protein downstream of mTORC1. Consistently, triciribine, an inhibitor of the Akt pathway, suppressed PDGF-BB-induced Akt phosphorylation without having any effect on S6 phosphorylation. Thus, mTORC2 does not appear to be upstream of mTORC1. We could also demonstrate that in Rictor-null cells the phosphorylation of phospholipase Cγ1 (PLCγ1) and protein kinase C (PKC) was impaired, and the PKCα protein levels strongly reduced. Furthermore, interfering with the PLCγ/Ca Platelet-derived growth factor (PDGF) stimulates proliferation, migration and survival of mesenchymal cells and plays a pivotal role during embryonic development and wound healing . The biomTOR is the mammalian ortholog of the yeast serine/threonine kinase TOR which is involved in the regulation of various cellular functions, such as initiation of translation, cell growth and proliferation, ribosome biogenesis, transcription and cytoskeletal reorganization . Dysregu3 that can interact with and thereby translocate Akt to the plasma membrane, where it is activated by phosphorylation on Ser473 in a hydrophobic motif and Thr308 in the activation loop of the kinase domain thymidine (0.1 μCi/ml). Incorporation of 3H-radioactivity into acid-insoluble material was measured by a scintillation counter. The obtained count per minute values in triplicate was normalized against the positive control of cultures incubated in 10% bovine serum for each experiment.For thymidine incorporation assay subconfluent cell cultures were serum-starved in 24-well plates and then incubated for 24 h in the presence or absence of rapamycin with PDGF-BB in DMEM containing [2. Cells adhering to the bottom of the filter were fixed by a 3-min incubation in 96% ethanol. The adherent cells were stained with Giemsa (Sigma)-and the migration indices were assessed by scanning the filter in a CCD camera (Fuji). Quantifications were performed using Aida Image Analyzer software. All experiments were performed in quadruplicate, and single representative data of three separate experiments ± SD are shown.Cell migration was determined as previously described . In brieErk: Extracellular regulated kinase; MAPK: Mitogen activated protein kinase; MEF: Mouse embryo fibroblast; mTOR: Mammalian target of rapamycin; PDGF: Platelet-derived growth factor; PDGFR: PDGF receptor; PLCγ1: Phospholipase C gamma 1; PI3K: Phosphatidylinositol 3-kinase.The authors declare that they have no competing interests.MR, planned and performed experiments, analyzed the data and contributed to manuscript preparation; CHH, planned experiments, analyzed data and contributed to manuscript preparation; JL, planned and performed experiments, analyzed the data and contributed to manuscript preparation. All authors read and approved the final manuscript.


Der p 7 is an important house dust mite allergen. However, antigenic determinants of Der p 7 are largely unknown. The purpose of this study is to analyze the determinants of Der p 7 and determine the structural basis of interactions between Der p 7 and WH9, an IgE-binding inhibition mouse monoclonal antibody (MoAb). IgE and WH9-reactive determinant(s) was identified by immunoblot using allergen mutants. A 3-D binary complex structure of Der p 7 and WH9 was simulated with homology modeling and docking methods. Our results obtained showed that among the five Der p 7 mutants , serum no. 1045 with IgE-binding against Der p 7 exhibited a reduced IgE immunoblot reactivity against Der p 7 L158A and D159A mutants. WH9 showed reduced immunoblot reactivity against S156A, L158A, D159A and P160A and the observation was confirmed by immunoblot inhibition. The WH9-binding determinant on Der p 7 containing S156, L158, D159 and P160 assumes a loop-like structure. The structural model of the Der p 7-WH9 complex suggests residues S156, I157, L158, D159 and P160 of Der p 7 contribute to WH9 binding via potential hydrogen bonds, electrostatic and hydrophobic interactions. In conclusion, MoAb WH9 interacts with critical residues L158 and D159 of Der p 7 and inhibits IgE-binding to Der p 7. Results obtained advance our understanding on molecular and structural bases of the antigenicity of Der p 7, its interactions with MoAb WH9 and facilitate the design of safer immunotherapy of human atopic disorders. There is a worldwide increase in the prevalence of human atopic disorders. Allergens cross-link mast cell-bound IgE antibodies can trigger a cascade of inflammatory and hypersensitive reactions. Characterization of IgE-binding determinants on allergens and delineation of the interaction modes between allergens and their specific antibodies at molecular and structural levels will enhance our understanding in disease mechanisms and development of effective therapeutic strategies towards these annoying human diseases.We have identified and characterized the important group 7 allergens including Der p 7 and Der f 7 which share 86% amino acid sequence identity and induce IgE antibodies in about 50% of mite-sensitized asthmatic patients Results from x-ray diffraction analysis of crystals containing allergen-IgE complexes can provide interacting details between allergens and IgE molecules. However, human IgE antibodies are polyclonal, their serum levels are low and their amino acid sequences are difficult to obtain. Therefore, even though the crystallographic structures of more than 50 allergens have been elucidated Recently, we determined the IgE-binding determinant(s) of Der f 7. We demonstrated that Asp 159 is a critical core residue for IgE-binding and contributes to IgE-mediated cross-reactivity between Der f 7 and Der p 7 In this study, we determined experimentally the Der p 7 antigenic determinants recognized by human IgE and MoAb WH9. We sequenced the variable regions of the heavy (VH) and light (VL) chains of WH9 and generated a structural model for the variable regions of this MoAb by homology modeling. Finally, we undertook molecular docking Sera (nos. 1045 and 1077) containing IgE antibodies against the group 7 dust mite allergens were collected from patients with a clinical history of bronchial asthma and stored in aliquots at −70°C until use 2 atmosphere. Culture supernatants containing MoAb WH9  were collected and stored in aliquots at −20°C. Antibody activity of the culture supernatants against the group 7 mite allergens was analyzed by immunoblotting. Culture supernatants containing MoAb HD19 against group 7 mite allergens Hybridoma WH9 E. coli JM109 for recombinant proteins expression. The mutations on the plasmids were confirmed by DNA sequencing and the recombinant proteins were affinity-purified with Ni-NTA resin columns (Qiagen) according to the manufacturer's instructions.Der p 7 mutants carrying single alanine substitute at S156, I157, L158, D159 or P160 were prepared essentially as described Reactivities of purified recombinant proteins against human IgE and mouse MoAbs were analyzed by SDS-PAGE-immunoblotting essentially as described For immunoblot inhibition studies, the culture supernatant from hybridoma WH9 was firstly incubated with 5 µg of purified recombinant wild-type Der p 7 or Der p 7 mutants at room temperature for 2.5 h before incubating with PVDF blots containing purified Der p 7 at room temperature for 1 h. As a control, the same culture supernatant from hybridoma WH9 was pre-incubated with 5 µg of bovine serum albumin  before immunoblotting against Der p 7.http://www. ncbi.nlm.nih.gov/BLAST) program while homologous alignment of sequences were performed with the CLUSTAL14 program. The complementarity determinant regions (CDRs) of WH9 variable domains were defined essentially according to the Kabat system Total RNA from hybridoma WH9 was isolated using a Trizol-reagent  according to the manufacturer's instructions. cDNAs encoding the heavy and the light chains of MoAb WH9 were obtained by reverse transcription with an AffinityScript Multiple Temperature cDNA Synthesis Kit . Sequencing of heavy chain and light chain variable regions were performed with PCR using primers listed in Protein homology modeling was performed essentially as described 2) on the backbone atoms, then on the Cα atoms, followed by gradually removing the constraints. A further 5×105 steps of minimization were performed to obtain an optimal Der p 7-WH9 complex structure for further analysis.Docking of the modeled structure of WH9 and the crystallographic structure of Der p 7 (PDB ID: 3H4Z) 151HIGGLSILDPIFGVL165) and the corresponding Der p 7 peptide Dp7-16 (151HIGGLSILDPIFAVL165) inhibited IgE binding to Der f 7 in serum nos. 990 and 1045 of asthmatic patients in a dot-blot inhibition assay In our previous study, Der f 7 peptide Df7-16  and the light (GenBank accession no. KC222649) chains of WH9 were obtained through PCR amplification of WH9 cDNA and summarized in We modeled the binary complex of Der p 7 and WH9 via computational docking. The Der p 7 model in The potential interactions between Der p 7 and WH9 are shown in Of the three CDRs on the heavy chain of WH9, two (CDR-H2 and CDR-H3) are in contact with Der p 7 . The sid2 atom of N31 on CDR-L1 makes two hydrogen bonds with Oδ1 and Oδ2 of D159 on Der p 7. In addition, the side-chain hydroxyl of Y32 of CDR-L1 forms two hydrogen bonds with the amide nitrogen of S156 and Oδ2 of D159 on Der p 7. Furthermore, the main-chain carbonyl oxygen of K96 forms a hydrogen bond with the amide nitrogen of D159 on Der p 7. The two Cγ atoms of V98 make two hydrophobic interactions with the Cδ atom of L158 on Der p 7. In addition, the Nζ of K96 forms an electrostatic network among the Oε1 and Oε2 of E97 on CDR-L3  and is similar to that of Ig-based antibodies and protein antigens (1400–2300 Å2) 2) We have validated our model by measuring the contact area between the antibody and the epitope. Our Der p 7-WH9 complex has a contact area of 1292.9 ÅIn summary, we characterized the antigenic determinants of Der p 7 through in vitro mutagenesis experiments and delineated its interactions with an IgE-binding inhibition MoAb WH9 through in silico antibody structure modeling and computational docking. In the absence of available X-ray deflection models, our study provides an alternative and powerful approach to characterize allergenic epitopes and demonstrates possible interactions between allergens and their specific antibodies at molecular level. Allergen-specific, IgE-binding-inhibition antibodies have been considered and tested in specific passive immunotherapy


Phosphorylation of the major photosynthetic light harvesting antenna proteins by STN7 kinase balances excitation between PSII and PSI. Phosphorylation of such abundant proteins is unique, differing distinctively from conventional tasks of protein kinases in phosphorylation of low abundance proteins in signaling cascades. Excitation balance between PSII and PSI is critical for redox homeostasis between the plastoquinone and plastocyanin pools and PSI electron acceptors, determining the capacity of the thylakoid membrane to produce reactive oxygen species (ROS) that operate as signals relaying information between chloroplasts and other cellular compartments. STN7 has also been proposed to be a conventional signaling kinase, instigating the phosphorylation cascade required for coordinated expression of photosynthesis genes and assembly of the photosynthetic machinery. The absence of STN7 kinase, however, does not prevent plants from sensing redox imbalance and adjusting the stoichiometry of the photosynthetic machinery to restore redox homeostasis. This suggests that STN7 is not essential for signaling between the chloroplast and the nucleus. Here we discuss the evolution and functions of the STN7 and other thylakoid protein kinases and phosphatases, and the inherent difficulties in analyzing signaling cascades initiated from the photosynthetic machinery. Based on our analyses of literature and publicly available expression data, we conclude that STN7 exerts it signaling effect primarily by controlling chloroplast ROS homeostasis through maintaining steady-state phosphorylation of the light harvesting II proteins and the redox balance in the thylakoid membrane. ROS are important signaling molecules with a direct effect on the development of jasmonate, which in turn relays information out from the chloroplast. We propose that thylakoid membrane redox homeostasis, regulated by SNT7, sends cell-wide signals that reprogram the entire hormonal network in the cell. Reversible protein phosphorylation modulates the cellular activity of target proteins by altering their chemical and structural characteristics, and is a ubiquitous mechanism for transmitting signals throughout the cell. Phosphorylation signal cascades usually involve the sequential phosphorylation and/or dephosphorylation of low abundance proteins including kinases, phosphatases, and protein receptors, culminating in the regulation of countless cellular metabolic processes. The reversible phosphorylation of several proteins in the plant thylakoid membrane is atypical, as these are among the most abundant membrane proteins in nature, and the physiological importance of this phenomenon is not yet fully understood. Phosphorylation of thylakoid proteins is known to play a role in coordinating the assembly and repair of the photosynthetic machinery, and in maintaining redox balance in the photosynthetic electron transfer chain (ETC) under changing environmental conditions. Membrane protein phosphorylation is also implicated in the transmission of retrograde signals from the thylakoid in response to the redox state of the intersystem ETC. This phosphorylation-mediated signal cascade has been suggested as a means of regulating the expression of chloroplast- and nuclear-encoded photosynthetic genes in response to environmental cues, hypothetically linking the operational status of the photosynthetic machinery with its assembly, repair, and acclimation according to environmental cues. The existence of such a retrograde mechanism remains hypothetical and any involvement of membrane phosphorylation, either directly or indirectly, is unclear.in silico analysis of their expression and evolution in plants. Our analysis describes the far-reaching impact of thylakoid protein phosphorylation on cell signaling, plant growth, and stress response that arises from a primary role of the kinases/phosphatases in safeguarding photosynthetic redox homeostasis and production of reactive oxygen species (ROS).At least 15 kinases and 10 phosphatases exist in higher plant chloroplasts  and Ostreococcus lucimarinus but not in red algae or diatoms , which occurs only at later stages of plant development. Strong expression of the thylakoid kinases and phosphatases, particularly STN8, in cotyledons may relate to their importance in assembly of PSII during early plant development. Additionally, they may be active in customizing the photosynthetic machinery for effective utilization of light energy during these early stages when the seedling is not capable of handling large amounts of reducing power. In light of this conceivably vital role during early plant development, it is puzzling that the stn8 and stn7/stn8 mutants lack any seedling phenotype compared to WT plants, although it is now clear that STN8 activity becomes important at moderate high light intensities  that occur in nature but are lower than the light intensities commonly used in laboratory high light experiments. Interestingly, recent analysis of the stn8 mutation in rice has described a stunted growth phenotype with severe problems in the degradation of damaged D1 protein .Unlike stn7 and psad mutants  mutants have been demonstrated to have inhibited chloroplast-to-nucleus signaling  mutant was included to elucidate chloroplast ROS-initiated and EXECUTER-protein-mediated signaling from the chloroplast . Furthermore the regulation of these genes was similar in both mutants  in the flu system. In flu mutants, dark-induced accumulation of chlorophyll intermediates causes a massive release of singlet oxygen upon re-illumination, inducing strong expression of a specific set of ROS-responsive genes , while the same genes are upregulated in high light and strongly upregulated in flu  and upregulated in psad  and stn7 . Based on the findings described above, we suggest that the redox state of the thylakoid membrane determines ROS production, which in turn induces oxylipin signals that are transmitted by EXECUTERs out of the chloroplast  signaling pathways  mutant compared to MeJA-treated WT  biosynthesis pathway and upregulates the EXECUTER (EX1 and EX2) proteins proposed to mediate emission of ROS signals from the chloroplast. We propose that the signaling molecules mediated by EX1 and EX2 proteins are jasmonates produced from thylakoid lipids according to the photosynthetic ROS status, by the enzymes that are strongly downregulated in stn7 and psad (Figure In d Figure . DisruptReactive oxygen species signals impact hormone signaling pathways that operate at all levels of cellular metabolism, both through direct interaction and via JA. Under standard growth conditions, the effects of ETC redox imbalance on hormone regulation appear to be buffered or counteracted by overlapping and redundant signaling pathways. It is unknown, however, how these readjustments to redox imbalance may compromise a plant’s ability to defend against plant pathogens, which is particularly relevant in the natural environment that presents continuous fluctuations in environmental conditions and an array of other abiotic and biotic stresses.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Interestingly, evidence has accumulated suggesting a role for S727 mono-phosphorylated STAT3. Here we show that latent KSHV infection of primary human endothelial cells (ECs) in vitro activates STAT3, and identify a key latency protein, kaposin B, that contributes to this activation. Kaposin B expression in ECs causes STAT3 phosphorylation at S727, in the absence of significant Y705 phosphorylation, and enhanced expression of a subset of STAT3 target genes including CCL5. Recent work shows that the tripartite motif-containing protein 28  negatively regulates STAT3 by recruiting transcriptional silencing complexes. The repressive activity of TRIM28 is mediated by post-translational modifications and a key site in the regulation of repressor activity maps to S473. Phosphorylation of this residue disrupts the recruitment of transcriptional silencing complexes effectively deactivating the co-repressive function of TRIM28. Confocal microscopy and western blot analysis demonstrate phosphorylation of TRIM28 at S473 in KSHV latently infected and kaposin B expressing ECs. Taken together, our studies suggest kaposin B may contribute to tumourigenesis via constitutive activation of STAT3.Kaposi’s sarcoma associated herpesvirus (KSHV) in the etiological agent for 3 AIDS-related cancers: Kaposi’s sarcoma (KS), primary effusion lymphoma, and multicentric Castleman’s disease. The molecular mechanisms used by KSHV to induce cancer are incompletely understood. KS lesions harbor proliferating latently-infected endothelial cells (ECs), large numbers of inflammatory cells, and marked neoangiogenesis. Considered the major driving force in the development of KS, these KSHV-infected ECs elaborate a variety of pro-inflammatory and angiogenic factors that contribute to tumourigenesis. Considerable evidence has accumulated suggesting a critical role for activated signal transducer and activator of transcription-3 (STAT3) in malignant transformation. STAT3 is a latent transcription factor that upon activation, drives the expression of a number of genes involved in cell proliferation, survival, and immune responses. Canonical STAT3 activation occurs via phosphorylation of Y705, dimerization, and nuclear translocation, followed by phosphorylation of S727 for maximal transcriptional activity. Activated STAT3 has been observed in a variety of malignancies and has been shown to induce fibroblast transformation


Anopheles gambiae and to correct deleterious mutations in genetic diseases. We describe the creation of an extensive set of HE variants with novel DNA cleavage specificities using an integrated experimental and computational approach. Using computational modeling and an improved selection strategy, which optimizes specificity in addition to activity, we engineered an endonuclease to cleave in a gene associated with Anopheles sterility and another to cleave near a mutation that causes pyruvate kinase deficiency. In the course of this work we observed unanticipated context-dependence between bases which will need to be mechanistically understood for reprogramming of specificity to succeed more generally.Homing endonucleases (HEs) can be used to induce targeted genome modification to reduce the fitness of pathogen vectors such as the malaria-transmitting  Targeted gene correction requires site-specific DNA cleavage to initiate cellular programs for DNA break repair . A doublLAGLIDADG homing endonucleases (LHEs) a have seBoth experimental-directed evolution  and compAnopheles gambiae reproduction  was transformed with a pCcdB plasmid. The pCcdB plasmids used contained either two adjacent copies of the activated LIB4 version . A standard procedure for electrocompetent cell preparation was used to produce each pCcdB-containing strain.A bacterial screen for variants of I-AniI that cleave single and multiple base-pair substitutions was carried out as previously described ,33 with  version  of the I version . The opt version , but sho4, 1 mM CaCl2) and on a LB-agar, 100 µg/ml carbenicillin control plate. If the selection was a Round 1, the volumes plated were 1 µl on the control plate and the entire remainder of the selection (spun down and resuspended) on the selection plate. Plates are grown for ∼36 h at 30°C. For Round 2 selections, colonies were picked from the Round 1 selection plate, grown overnight, combined and miniprepped, and 1 µl of the mixed plasmid was re-transformed into 20 µl of the same competent cell line as the Round 1 ligation. The volumes plated on the Round 2 selection plates depended on the Round 1 survival and size of the starting library, but 0.2–1 µl was plated on the control plate and typically 0.2–1 µl and 200 µl on selection plates. Colonies on Round 2 selection plates were either grown up for a third round of selection or subjected to colony PCR to amplify the endonuclease ORF off of the pENDO-HE vector and the PCR products were sequenced.For Round 1 selections, endonuclease libraries were ligated into the new negative selection pENDO-HE vector (containing two copies of the LIB4 WT I-AniI site), purified and transformed into 20 µl of the appropriate pCcdB-containing cell line. Ligation products were purified by running over a PCR cleanup column, leaving the wash buffer on the column for an extended period of time (5 min) to remove salts, and eluting the samples in 10 µl of water, all of which was transformed. Transformed bacteria were recovered in Terrific Broth (TB) media at 37°C for approximately one-half hour. The recovery was followed by a selection in 2 ml of liquid culture, containing 0.02% L-arabinose and 100 µg/ml carbenicillin, for 4 h at 30°C. After liquid selection, a volume of the culture was plated on a minimal media selection plate  of linearized substrate for 30 min at 37°C. For every variant, eight serial 2-fold dilutions, ranging from 5 to 1500 nM of purified protein, were performed in 1.25X reaction buffer and then were diluted with substrate to 1X in a 10 µl volume. Reactions were halted with ∼17 nM EDTA, followed by 60°C incubation for 5–10 min. Cleavage products were separated on a 1.2% agarose TBE gel and stained with ethidium bromide. The spectral density of the substrate and product bands was quantified using ImageJ, as previously described. Fraction cleavage, calculated by dividing the total density of product and substrate bands by the product band densities, was plotted versus endonuclease concentration using GraphPad Prism. At least two independent assays were completed for each cleavage experiment. The EC1/2max was calculated by fitting the fraction cleavage to a sigmoid function and using a value of 1.5 for the Hill coefficient, as previously described  or with XbaI for the pCcdB targets . Additional substrates, used in the assays shown in escribed . Kineticr detail , except r detail  as the fAll protocols were implemented within the Rosetta molecular modeling package . They arSupplementary Tables S1 and S2.Our strategy for using amino acid sidechain-base interactions frequently observed in the protein databank—interaction ‘motifs’—was introduced in . These mA. gambiae genome and to produce a list of genes with potentially engineerable I-AniI target sites within their coding sequence, ranked in order of engineerability, to produce a subset of 500 genes. The code for the PSSM program that was used, written in C++ for rapid analysis of entire organism genomes, is available on github at the following address:A position-specific scoring matrix (PSSM) matrix was applied to interrogate the https://github.com/jashworth-isb/pssm–tau)   of this 5 or greater, as there would be on the order of 100 background colonies. To overcome these difficulties, we introduced an additional selective pressure for specificity that reduces background survival, and increased system throughput by including several target sites (>10) in tandem onto a single pCcdB plasmid, allowing a single bacterial strain to be used to test multiple targets.Bacterial selection can be a powerful tool for reprogramming protein–DNA interactions and screening computationally designed libraries. A previously described selection system couples survival of bacteria to cleavage of a plasmid containing both the target site of interest and an ORF coding for a toxic DNA gyrase poison (CcdB) . Target in vitro cleavage assays resulted in low bacterial survival when included on the pENDO-HE plasmid, while positions that are cleaved with low efficiency displayed high survival . Enzymes with high specificity for their intended target show high survival in a system with the WT site on pENDO-HE (Supplementary Methods). The improved system was used to engineer enzymes for multiple base-pair substitutions and full target sites in genes of interest.To select for enzyme specificity in addition to activity, a target DNA sequence  for which cleavage is not desired is placed on the plasmid expressing the endonuclease protein (pENDO-HE) so that its cleavage results in loss of the HE ORF and hence cell death due to CcdB expression. Bacterial survival is thus linked to cleavage of the desired site on the toxic pCcdB as well as lack of cleavage of the undesired site on the nuclease encoding pENDO-HE b. As exppENDO-HE b, indicaSupplementary Table S1, Supplementary Figure S2). Initially, libraries were made by randomizing several protein residues neighboring base-pairs for which no successful computational designs were previously recovered. In addition to generating variants using randomized libraries, several previously described I-AniI computational designs .The bacterial selection system with the negative selection specificity modification was used to generate highly specific variants of the LAGLIDADG endonuclease I-AniI that cleave sites with single base-pair substitutions in the WT target .For each substitution in the I-AniI target site, libraries were generated where an amino acid type computationally predicted to make a motif interaction was kept fixed and surrounding residues were varied. These libraries were built both for single base-pair substitutions for which variants had already been identified with the fully randomized approach, and also for positions were no variant had yet been identified. Endonuclease variants with new specificities were selected from these motif-derived libraries, including some for target site substitutions for which active nuclease variants were not previously recovered. The likelihood of identifying an active and specific variant is increased in these computational motif libraries because all members contain a predicted high-quality contact and because the library size is reduced . For exaSupplementary Figure S2 and Supplementary Table S1). There are 60 possible single base-pair substitutions in the 20 bp I-AniI site (20 × 3). In this work, we attempted to generate variants cleaving 39 of the 60 substitutions, excluding those under the two edge loops of the endonuclease . Libraries were attempted and no reasonably active variants were identified for −9A, −9T, −6A, −4A, +6A, +6T and all three substitutions at position −7. This extensive dataset of single base-pair specificity switches is a valuable benchmark that can be used for evaluating and guiding the improvement of computational design methods. We describe preliminary results comparing computational predictions and experimental data in the Supplementary Material .The combination of the selected variants described and those previously published ,34 allownterface ) and thonterface ,34,42,43nterface ,34 out oAnopheles reproduction . Ta. TaAnophoduction .Supplementary Figure S7). However, this approach is unlikely to work for base substitutions directly neighboring one another as mutating amino acids to alter the specificity at one position could result in changes in specificity at the neighboring bases. Also, if multiple variants have been identified for a particular single base-pair substitution, utilizing only one such variant does not take advantage of the sequences of others that may prove more compatible with other desired target site changes. To address these challenges, we combined information from the already identified I-AniI single base-pair variants with computational modeling, assessing the compatibility of the amino acid mutations present in the single-site cleaving variants and identifying new motif residues for adjacent substitutions, and performed further in vivo selection. We broke down the target sites into groups of substitutions that were close together or into half-sites, building separate libraries for groups of substitutions in the two half-sites.Redesign to cleave target sites of interest will almost always involve changing specificity at multiple base-pairs. In early experiments, we found that mutations from two single base-pair variants could be successfully combined without further selection, even when they were separated by only a single base-pair (Supplementary Table S2). The mutations identified by screening of this library using the bacterial selection system resulted in a variant with increased specificity at position-5 , and the WT specificity was maintained at positions −2 and +1 (Supplementary Table S2). Sequences of active endonucleases from these S166K selections were used to generate further libraries that incorporated randomization over the +7G nt. The evolved variant chosen from this selection cleaved +7G well, whereas the WT enzyme did not  . However, when these mutations were combined with those from several variants available that cleave +3C well , none of the libraries tested yielded endonucleases with high specificity and activity for the entire (+) half-site. This result suggests that there may be unanticipated coupling between regions of the endonuclease interface.While this step-wise approach proved successful for engineering variants cleaving both halves of the CPK2 site, we had less success for a target in the FAH1313 gene . We werekcat*) than the +3C target alone. Surprisingly, however, the triply substituted site +1G/+3C/+5G is cleaved very slowly (low kcat*). To determine whether this unexpected coupling is specific to the evolved variant, we measured the cleavage rates of the WT I-AniI endonuclease (Y2 version) on the same set of target substrates . The overall pattern of kcat*-values was very similar between the two enzymes , we generated a variant cleaving a site with two single base-pair substitutions present in both the AGAP004671 and AGAP003225 (+) half-sites. This variant cleaves the AGAP003225 (+) half-site with an in vitro EC1/2max of 22 nM and shows activity in human cell assays   ,46.Anopheles target sites also contain additional substitutions that are partially tolerated by the WT endonuclease  with a rate of 9.4 min−1  half-sites. We attempted to generate endonucleases to cleave these full-sites by combining the tested mutations for each half-site with no further engineering. Both the CPK2 and AGAP003225 sites were successfully cleaved a and b. .4 min−1 , but . Determining how to improve suboptimal enzymes would help to identify features of endonuclease function that are currently missing from our models.While our successes cleaving the CPK2 site and AGAP003225 site are promising, kinetic analysis of the CPK2 enzyme revealed that its binding affinity is suboptimal  and willWe have identified several challenges that will need to be resolved for robust rational redesign of endonuclease specificity, notably unexpected context dependencies between base-pair substitutions , 5 and 6Supplementary Data are available at NAR Online, including [61–69].National Science Foundation (NSF) (graduate research fellowship to S.B.T.); US National Institutes of Health (NIH) and Foundation for the NIH through the Gates Foundation Grand Challenges in Global Health Initiative ; Howard Hughes Medical Institute (HHMI). The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH. Funding for open access charge: NIH grants or HHMI funds (to D.B.).Conflict of interest statement. None declared.


Eumycetoma is a morbid chronic granulomatous subcutaneous fungal disease. Despite high environmental exposure to this fungus in certain regions of the world, only few develop eumycetoma for yet unknown reasons. Animal studies suggest that co-infections skewing the immune system to a Th2-type response enhance eumycetoma susceptibility. Since chronic schistosomiasis results in a strong Th2-type response and since endemic areas for eumycetoma and schistosomiasis do regionally overlap, we performed a serological case-control study to identify an association between eumycetoma and schistosomiasis. Compared to endemic controls, eumycetoma patients were significantly more often sero-positive for schistosomiasis , but not for toxoplasmosis, an infection inducing a Th1-type response . Here, we show that schistosomiasis is correlated to susceptibility for a fungal disease for the first time. Schistosoma species, than healthy controls from the same region. In contrast, eumycetoma patients did not have more often antibodies against Toxoplasma species. This might implicate that schistosomiasis predisposes eumycetoma development. If schistosomiasis indeed predisposes eumycetoma development, eradicating Schistosoma in a population could also lower the number of eumycetoma cases in that area, which in the end could lead to intervention strategies not only for schistosomiasis but also for eumycetoma.Eumycetoma is a mutilating fungal disease of mainly the foot and is found in (sub)tropical regions such as Sudan. At the moment it is not understood why some people develop eumycetoma and others not. In the regions were eumycetoma is prevalent many other infections are also found. These infections could alter the immune system which makes people more or less susceptible in obtaining another infection. One of the infections with such an effect is Schistosomiasis. In Africa, eumycetoma is found in regions were schistosomiasis is prevalent. In this study we show that eumycetoma patients more often have antibodies against  Madurella mycetomatis (eumycetoma) M. mycetomatis, and thus have been exposed to M. mycetomatis, only few of them actually developed eumycetoma Eumycetoma is a chronic granulomatous subcutaneous infectious disease endemic in many tropical and sub-tropical regions in the so-called mycetoma belt between 30°N and 15°S of the equator M. mycetomatis. Secondly, genetic polymorphisms in the host involved in sex hormone synthesis and neutrophil function have already been associated with eumycetoma, indicating that the genetic make-up of the host is a crucial factor in susceptibility to eumycetoma development M. mycetomatis. This possibility is supported by the observation that M. mycetomatis could only induce eumycetoma in animals in the presence of an adjuvant predisposing towards a Th2-response Multiple explanations can be considered for the scanty susceptibility to eumycetoma. Firstly, genetic differences in the pathogen might exist that could lead to pathogenic and non-pathogenic variants of Skewing of the immune response is highly affected by invasive pathogens Based upon the above mentioned observations, and the fact that we recently have shown that eumycetoma patients have increased concentrations of circulating IL-10 A total of 84 serum samples was taken from 53 eumycetoma patients and 31 controls, matched for age and gender, in the endemic areas of Sudan between 2001 and 2008 . Serum sToxoplasma gondii were determined with the commercially available Toxo IgG II assay on the automated Liaison serology platform according to the manufacturer's protocol . Antibody levels against Schistosoma species were determined as described before by a combination of a commercial indirect hemagglutination test with Schistosoma mansoni adult worm antigens  and an enzyme-linked immunosorbent assay with homemade S. mansoni Soluble Egg Antigens (SEA) Schistosoma spp. serology was only considered positive when a positive result was obtained in both the IHA and SEA-ELISA tests.Specific IgG antibodies against Madurella mycetomatis Translationally Controlled Tumour Protein (TCTP) were measured with Luminex Technology as described before Antibody levels against U test was used to compare differences between IgG levels raised against the MmTCTP antigen in the study populations (GraphPad Instat 3.00). The Kruskal-Wallis test (SPSS Inc 17) was used to test if concentrations of antibodies against Schistosoma spp. differed significantly between patients with larger lesions compared to patients with smaller lesions, by including size  as the grouping variable. A value of p<0.05 was considered significant.Difference in positive and negative serology for schistosomiasis and toxoplasmosis between eumycetoma patients and endemic controls were calculated with the Chi square test (GraphPad Instat 3.00) by determining both the two-sided p-value and the Odds Ratio using Yates correction. The 95% confidence interval of the Odds Ratio was calculated using the approximation of Woolf. The Mann-Whitney Between 2001 and 2008, 53 patients and 31 endemic controls were included in the study. Most patients were male and had eumycetoma of the foot . All patSchistosoma infections as compared to endemic controls . In other words, an association exists between the infections caused by Madurella mycetomatis and Schistosoma spp. since the odds ratio for co-occurring schistosomiasis in eumycetoma patients is 3.2 . In contrast, eumycetoma patients were not significantly more often sero-positive for Toxoplasma gondii infections . Since schistosomiasis is known to reduce the humoral immune response against co-infecting pathogens serology . This suHelicobacter pylori, Fasciola hepatica, Echinostoma and with Plasmodium in case of S. haematobium schistosomiasis Leishmania donovaniToxoplasma gondii, Entamoeba histolytica infections and Plasmodium in case of S. mansoni schistosomiasis Schistosomiasis is a chronic disease with an estimated 200 million people infected in subtropical countries Schistosoma spp.. As a control, Toxoplasma gondii infections were monitored since this infection is also endemic in Sudan and results in a Th1-type of immune response in immune-competent hosts Since eumycetoma infections can only be established in animals with adjuvants inducing a strong Th2-type immune response, we hypothesized that co-infections inducing a Th2-type immune response would predispose to eumycetoma disease. This study compared eumycetoma patients with matched endemic controls without eumycetoma for co-infections with Toxoplasma gondii in our study population was 42%, which was exactly the same as found by Abdel-Hameed et al. in Gezira in 1991 Toxoplasma, another prevalent infection in Sudan, did not correlate with eumycetoma disease. Furthermore no correlation was found between seropositivity of schistosomiasis and toxoplasmosis.Although we only studied a limited number of people, which might not represent the full population of the study area, we did find an overall sero-prevalence of schistosomiasis in our study population of 51%, which is consistent with the earlier reported variable sero-prevalences for schistosomiasis in Sudan in the New Halfa and Um Zukra villages in the Gezira and Kassala regions  (16% andNocardia brasiliensisM. mycetomatis eumycetoma patients in Sudan The main underlying cause may be the strong Th2-response induced by schistosomiasis and the high expression of interleukin-10 (IL-10) by Th2 cells In conclusion, even among this relatively small number of patients, eumycetoma was significantly associated with schistosomiasis and not with toxoplasmosis. Since this correlation is only based on serological data, animal studies are currently performed to investigate the precise role of schistosomiasis and Th2-predisposition in the development of fungal eumycetoma.Checklist S1STROBE checklist.(DOC)Click here for additional data file.


Prostaglandin E2 (PGE2) is a key regulator of immunopathology in inflammatory reactions. Little is known about the mechanisms of BisGMA-induced PGE2 expression in macrophage. The aim of this study was to evaluate the signal transduction pathways of BisGMA-induced PGE2 production in murine RAW264.7 macrophages.Bisphenol A-glycidyl-methacrylate (BisGMA) employs as a monomer in dental resins. The leakage of BisGMA from composite resins into the peripheral environment can result in inflammation Herein, we demonstrate that BisGMA can exhibit cytotoxicity to RAW264.7 macrophages in a dose- and time-dependent manner (p<0.05). In addition, PGE2 production, COX-2 expression, and cPLA2 phosphorylation were induced by BisGMA on RAW264.7 macrophages in a dose- and time-dependent manner (p<0.05). Moreover, BisGMA could induce the phosphorylation of ERK1/2 pathway , p38 pathway , and JNK pathway  in a dose- and time-dependent manner (p<0.05). Pretreatment with AACOCF3, U0126, SB203580, and SP600125 significantly diminished the phosphorylation of cPLA2, ERK1/2, p38, and JNK stimulated by BisGMA, respectively (p<0.05). BisGMA-induced cytotoxicity, cPLA2 phosphorylation, PGE2 generation, and caspases activation were reduced by AACOCF3, U0126, SB203580, and SP600125, respectively (p<0.05).via COX-2 expression, cPLA2 phosphorylation, and the phosphorylation of MAPK family. Cytotoxicity mediated by BisGMA may be due to caspases activation through the phosphorylation of cPLA2 and MAPKs family.These results suggest that BisGMA induced-PGE2 production may be  PGE2 is a metabolite of arachidonic acid (AA) and is progressively produced by cytosolic phospholipase A2 (cPLA2), cyclooxygenases (COX), and PG synthases via COX2 protein expression in human pulp cells Previously, BisGMA was found to generate PGE2 Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), streptomycin and penicillin were obtained from Life Technologies . Enhanced chemiluminescence reagents were purchased from GE Healthcare . PGE2 ELISA kit was obtained from eBiosciences . Antibodies for COX-2, non-phosphorylation types of p38, cPLA2, MEK1/2, ERK1/2, Elk, MEK3/6, MAPKAPK2, MEK4, JNK, cJUN, phosphorylation types of cPLA2 (Ser505), MEK1/2 (Ser218/Ser222), ERK1/2 (Tyr204), Elk (Ser383), MEK3/6 (Ser189/Ser207), MAPKAPK2 (Thr222), MEK4 (Ser80), JNK (Thr183/Tyr185), cJUN (Ser63/73), and arachidonyl trifluoromethyl ketone (AACOCF3) were obtained from Santa Cruz Biotechnology . Antibodies for the phosphorylation type of p38 (Thr180/Tyr182) was purchased from Cell Signaling Technology . Secondary antibodies were obtained from Jackson ImmunoResearch Laboratories . 1,4-di-amino-2,3- dicyano-1,4-bis [2-amino-phenylthio] butadiene (U0126), 4-(4-fluorophenyl)-2-(4-methylsulfinyl-phenyl)-5-(4-pyridyl)-1Himidazole (SB203580), and Anthra pyrazol-6(2H)-one (SP600125) were obtained from Calbiochem-Novabiochem . Other chemicals were purchased from Sigma-Aldrich . BisGMA was dissolved in dimethyl sulfoxide (DMSO). The final volume of DMSO added was lower than 0.5% which is a non-toxic concentration.2 humidified atmosphere at 37°C. To investigate the effects of BisGMA on RAW264.7 macrophages, cells were seeded on the plates and cultured for 24 h. After cell attachment, the medium were replaced with serum-free medium. Cells pretreated with or without 1 µM ERK1/2 inhibitor U0126, 30 µM p38 inhibitor SB203580, 30 µM JNK inhibitor SP600125, and 30 µM cPLA2 inhibitor AACOCF3 for 30 min. And then, RAW264.7 macrophages were incubated with different concentrations  of BisGMA for 15 min or 2 h.Murine macrophage cell line, RAW264.7, was obtained from Bioresource Collection and Research Center . Cells were cultured in DMEM containing 10% FBS, 100 µg/ml streptomycin, and 100 U/ml penicillin. RAW 264.7 cells were maintained at sub-confluence in a 95% air and 5% CO5 cells were treated with various concentrations  of BisGMA for the indicated treatment period. Then, 1% Triton-X 100 was added to another well and incubated for 45 min. 50 µl supernatants of each well was transferred to new 96-well plate and added 50 µl of reconstituted substrate mix from CytoTox® 96 nonradioactive assay kit . After 30 min, the stop solution was added to each well. Absorbance was recorded at 490 nm by using a microplate reader . Cell viability was calculated according to our perious study Cytotoxicity was measured by the release of lactate dehydrogenase (LDH). Briefly, 1×104 cells were incubated with different concentrations  of BisGMA for 2 h. The PGE2 levels in culture medium were measured by ELISA kit  according to the manufacturer’s instructions.The protein concentrations of PGE2 were measured by using ELISA To investigate the expression of COX2, phosphorylation and non-phosphorylation types of cPLA2, cells were treated with BisGMA for 2 h. To evaluate the phosphorylation and non-phosphorlation types of MEK1/2, ERK1/2, Elk, MEK3/6, p38, MAPKAPK2, MEK4, JNK, and cJUN, cells were treated with BisGMA for 15 min. After treatment, 50 µg of cell lysate protein, was separated by SDS-PAGE, and electrophoretically transferred to polyvinylidene difluoride membrane. The membranes were blocked with 5% (w/v) nonfat dried milk for 1 h at room temperature to reduce nonspecific binding, washed with PBS containing 0.1% Tween-20 (PBST), then probed with antibodies including COX-2, phosphorylation and non-phosphorylation of cPLA2, MEK1/2, ERK1/2, Elk, MEK3/6, p38, MAPKAPK2, MEK4, JNK, and cJUN. After membranes were washed again with PBST, a 1∶10,000 (v/v) dilution of horseradish peroxidase-labeled IgG was added at room temperature for 1 h, and the blots were developed using enhanced chemiluminescence reagents. The phosphorylation and expression of proteins were quantified with a LAS-4000 mini luminescent image analyzer  Caspases activities were analyzed with a caspases fluorometric assay kits  according to our recent study At least three independent experiments were performed as indicated in the figure legends. All data were expressed as mean ± standard deviation (SD). Statistical analysis was performed by using one-way analysis of variance (ANOVA) with Bonferroni-adjusted post hoc tests for multigroup comparisons; p<0.05 was considered significantly for each test.The changes of RAW264.7 macrophage morphology and cell density after exposure to 3 µM BisGMA for the indicated treatment periods and the indicated concentrations of BisGMA for 2 h were shown in The effect of BisGMA on the generation of PGE2 in RAW264.7 macrophages by ELISA assay was shown in The exposure of RAW264.7 macrophages to BisGMA evoked the phosphorylation of cPLA2 in a time-dependent manner (p<0.05) . The durPretreatment of AACOCF3 significantly diminished BisGMA-induced cPLA2 phosphorylation from 2.09 to 1.36 fold (p<0.05) . PretreaThe exposure of RAW264.7 cells to BisGMA evoked the phosphorylation of ERK1/2 in a time-dependent manner (p<0.05) . The durThe exposure of RAW264.7 cells to BisGMA evoked the phosphorylation of p38 in a time-dependent manner (p<0.05) . 3 µM BiThe exposure of RAW264.7 cells to BisGMA evoked the phosphorylation of JNK in a time-dependent manner (p<0.05) . The durPretreatment of U0126 significantly diminished BisGMA-stimulated phosphorylation of cPLA2 from 2.4 to 1.5 fold (p<0.05) . PretreaPretreatment of U0126, SB203580, SP600125, and AACOCF3 significantly diminished BisGMA-induced caspase 3 activation from 6289.07 to 1532.79, 1027.51, 1674.3, and 2038.10 RFU/µg lysate, respectively (p<0.05) . PretreaBisGMA is the most common used resin monomer for dental materials such as bonding agents, sealants, cement, bite guards, and dentures. To restore the caries cavity on the teeth bottom, the unpolymerized monomers of BisGMA can leach into oral cavity and prompt pulpal imflammation, cytoxicity, and delay pulpal healing via NFκB phosphorylation.PGE2 plays an important role in acute and chronic inflammation, infection, pain, fever, and cancer via the phosphorylation of cPLA2.cPLA2 catalyzes the first step in hydrolysis of membrane phosphatidylcholine convert into AA, which is the precursor of proinflammatory lipid mediators such as PGs, thromboxane, and leukotrienes RSK. p38 is activated and phosphorylated by MEK3/6. MEK3/6 is in turn regulated by MEKK1/2/3/4. The substrate of p38 is MAPKAPK2 and ATF2. JNK is activated and phosphorylated by MEK4/7. MEK4/7 is in turn regulated by MEKK1/2/3/4. The substrate of JNK is cJUN via MAPKs signal transduction pathways.Several studies have demonstrated the activation and phosphorylation of cPLA2 regulated by MAPKs in macrophage during stimulation via the generation of reactive oxygen species and the expression of hemeoxygenase-1 and carboxylesterase could against BisGMA-induced cytotoxicity in human pulp cells Recently, we demonstrated that BisGMA-induced cytotoxicity and apoptosis may be via caspases 3, 8, and 9 activation in RAW264.7 macrophages via COX-2 expression and cPLA2 phosphorylation. BisGMA induced the activation and phosphorylaion of p38 pathway, including MEK3/6, p38, and MAPKAPK2. BisGMA also induced the activation and phosphorylaion of ERK1/2 pathway, including MEK1/2, ERK1/2, and Elk. In addition, BisGMA induced the activation and phosphorylaion of JNK pathway, including MEK4, JNK, and cJUN. Moreover, U0126, SB203580, SP600125, and AACOCF3 inhibited BisGMA-induced cPLA2 phosphorylation, PGE2 generation, cytotoxicity, and caspases activation. In conclusion, we have shown that the generation of PGE2 and caspases activation induced by BisGMA may involve the phosphorylation of cPLA2 via ERK1/2, p38, and JNK pathways in RAW264.7 macrophages.This study has investigated the mechanism of PGE2 generation and cytotoxicity induced by BisGMA in RAW264.7 macrophages . The pri