Abstract
Lysophosphatidylcholines (lysoPCs) are effective polymorphonuclear neutrophil (PMN) priming agents implicated in transfusion-related acute lung injury (TRALI). LysoPCs cause ligation of the ...G2A receptor, cytosolic Ca2+ flux, and activation of Hck. We hypothesize that lysoPCs induce Hck-dependent activation of protein kinase C (PKC), resulting in phosphorylation and membrane translocation of 47 kDa phagocyte oxidase protein (p47phox). PMNs, human or murine, were primed with lysoPCs and were smeared onto slides and examined by digital microscopy or separated into subcellular fractions or whole-cell lysates. Proteins were immunoprecipitated or separated by polyacrylamide gel electrophoresis and immunoblotted for proteins of interest. Wild-type (WT) and PKCγ knockout (KO) mice were used in a 2-event model of TRALI. LysoPCs induced Hck coprecipitation with PKCδ and PKCγ and the PKCδ:PKCγ complex also had a fluorescence resonance energy transfer (FRET)+ interaction with lipid rafts and Wiskott-Aldrich syndrome protein family verprolin-homologous protein 2 (WAVE2). PKCγ then coprecipitated with p47phox. Immunoblotting, immunoprecipitation (IP), specific inhibitors, intracellular depletion of PKC isoforms, and PMNs from PKCγ KO mice demonstrated that Hck elicited activation/Tyr phosphorylation (Tyr311 and Tyr525) of PKCδ, which became Thr phosphorylated (Thr507). Activated PKCδ then caused activation of PKCγ, both by Tyr phosphorylation (Τyr514) and Ser phosphorylation, which induced phosphorylation and membrane translocation of p47phox. In PKCγ KO PMNs, lysoPCs induced Hck translocation but did not evidence a FRET+ interaction between PKCδ and PKCγ nor prime PMNs. In WT mice, lysoPCs served as the second event in a 2-event in vivo model of TRALI but did not induce TRALI in PKCγ KO mice. We conclude that lysoPCs prime PMNs through Hck-dependent activation of PKCδ, which stimulates PKCγ, resulting in translocation of phosphorylated p47phox.
Uncontrolled inflammatory responses underlie the etiology of acute lung injury and acute distress respiratory syndrome, the most common late complications in trauma, the leading cause of death under ...the age of 59. Treatment with HTS decreases lung injury in clinical trials, rat models of trauma and hemorrhagic shock and inflammation in lung cell lines, although the mechanisms underlying these responses are still incompletely understood. Transcriptomics (RNaseq), proteomics, and U-13C-glucose tracing metabolomics experiments were performed to investigate the mechanisms of cellular responses to HTS treatment in primary small airway epithelial cells in the presence or absence of inflammatory injury mediated by a cocktail of cytokines (10 ng/mL of IFNγ, IL-1β, and TNFα). Modestly hyperosmolar HTS has an anti-inflammatory effect, triggers the p53–p21 signaling axis, and deregulates mitochondrial metabolism while inducing minimal apoptosis in response to a second hit by cytokines. Decreased transcription of pro-inflammatory cytokines suggested a role for the tumor suppressor protein p53 in mediating the beneficial effects of the HTS treatment. The anti-inflammatory mechanisms induced by HTS involves p53 gene regulation, promotes cell cycle arrest, and prevents ROS formation and mitochondria depolarization. Pharmaceutical targeting of the p53–p21 axis may mimic or reinforce the beneficial effects mediated by HTS when sustained hypertonicity cannot be maintained.
Introduction
Tissue injury (TI) and hemorrhagic shock (HS) are the major contributors to trauma-induced coagulopathy (TIC). However, the individual contributions of these insults are difficult to ...discern clinically because they typically coexist. TI has been reported to release procoagulants, while HS has been associated with bleeding. We developed a large animal model to isolate TI and HS and characterize their individual mechanistic pathways. We hypothesized that while TI and HS are both drivers of TIC, they provoke different pathways; specifically, TI reduces time to clotting, whereas, HS decreases clot strength stimulates hyperfibrinolysis.
Methods
After induction of general anesthesia, 50 kg male, Yorkshire swine underwent isolated TI (bilateral muscle cutdown of quadriceps, bilateral femur fractures) or isolated HS (controlled bleeding to a base excess target of − 5 mmol/l) and observed for 240 min. Thrombelastography (TEG), calcium levels, thrombin activatable fibrinolysis inhibitor (TAFI), protein C, plasminogen activator inhibitor 1 (PAI-1), and plasminogen activator inhibitor 1/tissue-type plasminogen activator complex (PAI-1-tPA) were analyzed at pre-selected timepoints. Linear mixed models for repeated measures were used to compare results throughout the model.
Results
TI resulted in elevated histone release which peaked at 120 min (
p
= 0.02), and this was associated with reduced time to clot formation (R time) by 240 min (
p
= 0.006). HS decreased clot strength at time 30 min (
p
= 0.003), with a significant decline in calcium (
p
= 0.001). At study completion, HS animals had elevated PAI-1 (
p
= 0.01) and PAI-1-tPA (
p
= 0.04), showing a trend toward hyperfibrinolysis, while TI animals had suppressed fibrinolysis. Protein C, TAFI and skeletal myosin were not different among the groups.
Conclusion
Isolated injury in animal models can help elucidate the mechanistic pathways leading to TIC. Our results suggest that isolated TI leads to early histone release and a hypercoagulable state, with suppressed fibrinolysis. In contrast, HS promotes poor clot strength and hyperfibrinolysis resulting in hypocoagulability.
BackgroundGlypican 1 (GPC1) is a heparan sulphate proteoglycan cell membrane protein. It is implicated in driving cancers of the breast, brain, pancreas, and prostate; however, its role in ...esophagogastric cancer (EGAC) remains unexplored. The aim of the study was to investigate and elucidate the molecular mechanistic of GPC1 in human EGAC. MethodsThirty tissue and 120 microarray sections of EGAC were evaluated with Anti-GPC1 immunohistochemistry. Loss and gain of GPC1 function were performed using lentivirus transfection in EGAC cell lines. Mechanistically, AKT/GSK/β-catenin pathway was evaluated using AKT inhibitor MK-2206 and Wnt/β-catenin stimulant LiCl. ResultsGPC1 overexpression was found in 102 cases (68%). Overexpression of GPC1 correlated with lymph node metastasis, poor differentiation and decreased overall survival. Lentivirus mediated GPC1 knockdown resulted in decreased cell proliferation, migration, invasion, and colony formation. Knockdown caused G0/G1 cell cycle arrest, increased apoptosis, and reduced epithelial mesenchymal transition (EMT). GPC1 mediated its effects by activation of AKT/GSK/β-catenin pathway. ConclusionsThis is the first descriptive study to decipher the role of GPC1 in EGAC. Our results suggest that GPC1 regulates cell proliferation and growth and may serve as an attractive oncotarget in EGAC.
Protein kinase C (PKC)α/β dependent signaling events downstream of TLR4 or TLR2 were investigated in neutrophils stimulated with LPS or PGN. Pretreatment of neutrophils with the structurally distinct ...PKCα/β inhibitors Gö6976 or GF109203X decreased nuclear translocation of NF-κB and production of the proinflammatory cytokine TNF-α. Inhibition of PKCα/β also prevented LPS or PGN induced phosphorylation of IKKα/β, phosphorylation and degradation of IκB-α, as well as phosphorylation of the p65 subunit of NF-κB. Activation of p38, JNK, and ERK 1/2 in response to TLR2 engagement was diminished in neutrophils in which PKCα/β was inhibited. However, no alteration in the activation of these kinases was found in TLR4 stimulated neutrophils when PKCα/β was blocked. Such results indicate that distinct intracellular signalling pathways leading to MAPK activation are induced by TLR4 and TLR2 stimulation. PKCα/β can regulate NF-κB dependent transcription in neutrophils both by enhancing nuclear translocation of NF-κB and also by stimulating phosphorylation of the p65 subunit.
Many receptors involved with innate immunity activate the inhibitor kappa B kinase signalosome (IKK). The active complex appears to be assembled from the two kinase units, IKKα and IKKβ with the ...regulatory protein NEMO. Because we previously found that RNA silencing of clathrin heavy chains (CHC), in transformed human lung pneumocytes (A549), decreased TNFα‐induced signaling and phosphorylation of inhibitor kappa B (IκB), we hypothesized that CHC forms cytoplasmic complexes with members of the IKK signalosome. Widely available antibodies were used to immunoprecipitate IKKα and NEMO interactomes. Analysis of the affinity interactomes by mass spectrometry detected clathrin with both baits with high confidence. Using the same antibodies for indirect digital immunofluorescence microscopy and FRET, the CHC–IKK complexes were visualized together with NEMO or HSP90. The natural variability of protein amounts in unsynchronized A549 cells was used to obtain statistical correlation for several complexes, at natural levels and without invasive labeling. Analyses of voxel numbers indicated that: (i) CHC–IKK complexes are not part of the IKK signalosome itself but, likely, precursors of IKK–NEMO complexes. (ii) CHC–IKKβ complexes may arise from IKKβ–HSP90 complexes.
Clathrin forms complexes with IKKa, IKKb, and NEMO, but apparently not the canonical signalosome. These complexes are identified, for the first time, by affinity proteomics and triple FRET without altering molecular structure.
Protein kinase C (PKC)alpha/beta dependent signaling events downstream of TLR4 or TLR2 were investigated in neutrophils stimulated with LPS or PGN. Pretreatment of neutrophils with the structurally ...distinct PKCalpha/beta inhibitors Go6976 or GF109203X decreased nuclear translocation of NF-kappaB and production of the proinflammatory cytokine TNF-alpha. Inhibition of PKCalpha/beta also prevented LPS or PGN induced phosphorylation of IKKalpha/beta, phosphorylation and degradation of IkappaB-alpha, as well as phosphorylation of the p65 subunit of NF-kappaB. Activation of p38, JNK, and ERK 1/2 in response to TLR2 engagement was diminished in neutrophils in which PKCalpha/beta was inhibited. However, no alteration in the activation of these kinases was found in TLR4 stimulated neutrophils when PKCalpha/beta was blocked. Such results indicate that distinct intracellular signalling pathways leading to MAPK activation are induced by TLR4 and TLR2 stimulation. PKCalpha/beta can regulate NF-kappaB dependent transcription in neutrophils both by enhancing nuclear translocation of NF-kappaB and also by stimulating phosphorylation of the p65 subunit.
Urokinase plasminogen activator (uPA) is a serine protease that catalyzes the conversion of plasminogen to plasmin. In addition, uPA has been shown to have proinflammatory properties, particularly in ...potentiating lipopolysaccharide (LPS)-induced neutrophil responses. To explore the mechanisms by which uPA exerts these effects, we examined the ability of specific uPA domains to increase cytokine expression in murine and human neutrophils stimulated with LPS. Whereas the addition of intact uPA to neutrophils cultured with LPS increased mRNA and protein levels of interleukin-1beta, macrophage-inflammatory protein-2, and tumor necrosis factor alpha, deletion of the kringle domain (KD) from uPA resulted in loss of these potentiating effects. Addition of purified uPA KD to LPS-stimulated neutrophils increased cytokine expression to a degree comparable with that produced by single-chain uPA. Inclusion of the arginine-glycine-aspartic but not the arginine-glycine-glutamic peptide to neutrophil cultures blocked uPA kringle-induced potentiation of proinflammatory responses, demonstrating that interactions between the KD and integrins were involved. Antibodies to alpha(V) or beta(3) integrins or to the combination of alpha(V)beta(3) prevented uPA kringle-induced enhancement of expression of proinflammatory cytokines and also of adhesion of neutrophils to the uPA KD. These results demonstrate that the KD of uPA, through interaction with alpha(V)beta(3) integrins, potentiates neutrophil activation.
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