ABSTRACT
Current evidence indicates that dysregulation of the host inflammatory response to infectious agents is central to the mortality of patients with sepsis. Strategies to block inflammatory mediators such as PAF have been investigated as adjuvant therapies for sepsis. PAF-AH, the enzyme responsible for PAF degradation, showed positive results in pre-clinical studies and phase II clinical trials, but the results of a phase III study were disappointing. In this study, we investigated the potential protective mechanism of PAF-AH in sepsis using the murine model of cecal ligation and puncture (CLP). Treatment with rPAF-AH increased peritoneal fluid levels of the anti-inflammatory mediators MCP-1/CCL2 after CLP. The numbers of bacteria (CFU) in the peritoneal cavity were decreased in the rPAF-AH-treated group, indicating more efficient bacterial clearance after rPAF-AH treatment. Interestingly, we observed increased levels of nitric oxide (NO) after PAF-AH administration, and rPAF-AH treatment did not decrease CFU numbers either in iNOS-deficient mice or in CCR2-deficient mice. We concluded that administration of exogenous rPAF-AH reduced inflammatory injury, altered cytokine levels and favored bacterial clearance with a clear impact on mortality through modulation of MCP-1/CCL2 and NO levels in a clinically relevant sepsis model.
Subject(s)
1-Alkyl-2-acetylglycerophosphocholine Esterase/administration & dosage , Sepsis/drug therapy , Sepsis/microbiology , Animals , Chemokine CCL2/biosynthesis , Disease Models, Animal , Humans , Male , Mice , Nitric Oxide/biosynthesis , Peritoneal Cavity/microbiology , Recombinant Proteins/administration & dosage , Salmonella Infections/drug therapy , Salmonella Infections/microbiology , Salmonella typhimurium , Sepsis/metabolismABSTRACT
Diacylglycerol (DAG) is an important lipid second messenger. DAG signalling is terminated by conversion of DAG to phosphatidic acid (PA) by diacylglycerol kinases (DGKs). The neuronal synapse is a major site of DAG production and action; however, how DGKs are targeted to subcellular sites of DAG generation is largely unknown. We report here that postsynaptic density (PSD)-95 family proteins interact with and promote synaptic localization of DGKι. In addition, we establish that DGKι acts presynaptically, a function that contrasts with the known postsynaptic function of DGKζ, a close relative of DGKι. Deficiency of DGKι in mice does not affect dendritic spines, but leads to a small increase in presynaptic release probability. In addition, DGKι-/- synapses show a reduction in metabotropic glutamate receptor-dependent long-term depression (mGluR-LTD) at neonatal (â¼2 weeks) stages that involve suppression of a decrease in presynaptic release probability. Inhibition of protein kinase C normalizes presynaptic release probability and mGluR-LTD at DGKι-/- synapses. These results suggest that DGKι requires PSD-95 family proteins for synaptic localization and regulates presynaptic DAG signalling and neurotransmitter release during mGluR-LTD.
Subject(s)
Brain/metabolism , Diacylglycerol Kinase/analysis , Diacylglycerol Kinase/metabolism , Nerve Tissue Proteins/metabolism , Receptors, Metabotropic Glutamate/metabolism , Synapses/metabolism , Animals , Brain/ultrastructure , Cell Line , Cells, Cultured , Diacylglycerol Kinase/genetics , Dizocilpine Maleate/metabolism , Gene Deletion , Gene Expression , Humans , Mice , Neurons/metabolism , Neurons/ultrastructure , Neurotransmitter Agents/metabolism , Protein Kinase C/metabolism , Rats , Rats, Sprague-Dawley , Receptors, N-Methyl-D-Aspartate/metabolism , Synaptic TransmissionABSTRACT
COX-2 promotes colon cancer. While both nonselective NSAIDs and selective COX-2 inhibitors reduce disease burden, their adverse gastrointestinal and cardiovascular side effects limit their therapeutic use. In this issue of the JCI, Zhang et al. used gene silencing and a derivative of licorice root to show that inhibition of the enzyme 11beta-hydroxysteroid dehydrogenase type II(11betaHSD2) reduces tumor COX-2 activity, tumor growth, and metastasis by increasing the tonic glucocorticoid-mediated suppression of the COX-2 signaling pathway without the adverse effects associated with NSAIDs and selective COX-2 inhibitors (see the related article beginning on page 876). Their findings suggest that 11betaHSD2 inhibition may be a potential therapeutic option in colon cancer, warranting further investigation.
Subject(s)
Colonic Neoplasms/drug therapy , Glycyrrhiza , 11-beta-Hydroxysteroid Dehydrogenase Type 2/antagonists & inhibitors , Animals , Anti-Inflammatory Agents, Non-Steroidal/adverse effects , Anti-Inflammatory Agents, Non-Steroidal/therapeutic use , Colonic Neoplasms/enzymology , Cyclooxygenase 2 Inhibitors/adverse effects , Cyclooxygenase 2 Inhibitors/therapeutic use , Enzyme Inhibitors/therapeutic use , Humans , Hydrocortisone/metabolism , Mice , Phytotherapy , Plant Extracts/therapeutic useABSTRACT
Diacylglycerol (DAG) is an important lipid signalling molecule that exerts an effect on various effector proteins including protein kinase C. A main mechanism for DAG removal is to convert it to phosphatidic acid (PA) by DAG kinases (DGKs). However, it is not well understood how DGKs are targeted to specific subcellular sites and tightly regulates DAG levels. The neuronal synapse is a prominent site of DAG production. Here, we show that DGKzeta is targeted to excitatory synapses through its direct interaction with the postsynaptic PDZ scaffold PSD-95. Overexpression of DGKzeta in cultured neurons increases the number of dendritic spines, which receive the majority of excitatory synaptic inputs, in a manner requiring its catalytic activity and PSD-95 binding. Conversely, DGKzeta knockdown reduces spine density. Mice deficient in DGKzeta expression show reduced spine density and excitatory synaptic transmission. Time-lapse imaging indicates that DGKzeta is required for spine maintenance but not formation. We propose that PSD-95 targets DGKzeta to synaptic DAG-producing receptors to tightly couple synaptic DAG production to its conversion to PA for the maintenance of spine density.
Subject(s)
Dendritic Spines/metabolism , Diacylglycerol Kinase/metabolism , Intracellular Signaling Peptides and Proteins/metabolism , Isoenzymes/metabolism , Membrane Proteins/metabolism , Synapses/metabolism , Synaptic Transmission/physiology , Animals , Cells, Cultured , Dendritic Spines/ultrastructure , Diacylglycerol Kinase/genetics , Diglycerides/metabolism , Disks Large Homolog 4 Protein , Guanylate Kinases , Humans , Intracellular Signaling Peptides and Proteins/genetics , Isoenzymes/genetics , Male , Membrane Proteins/genetics , Mice , Mice, Knockout , Neurons/cytology , Neurons/metabolism , Patch-Clamp Techniques , Protein Kinase C/antagonists & inhibitors , Protein Kinase C/metabolism , Rats , Rats, Sprague-Dawley , Synapses/ultrastructureABSTRACT
We generated mice expressing a COX-2 transgene in colon epithelium and found that they did not develop spontaneous colon tumors. But when treated with azoxymethane, a colon carcinogen, COX-2 mice had a higher tumor load compared to wild-type mice. There was no change in the number of pre-neoplastic lesions, indicating that COX-2 does not affect tumor initiation. Tumors in the COX-2 transgenic mice had higher levels of phosphorylated epidermal growth factor receptor and Akt compared to wild-type mice. Collectively, our data indicate that COX-2 promotes colon tumor progression, but not initiation, and it does so, in part, by activating EGFR and Akt signaling pathways.
Subject(s)
Cyclooxygenase 2/genetics , Epithelium/metabolism , ErbB Receptors/metabolism , Gene Expression Regulation, Neoplastic , Intestinal Mucosa/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Transgenes , Animals , Cyclooxygenase 2/biosynthesis , Disease Progression , Female , Humans , Male , Mice , Mice, Transgenic , Signal TransductionABSTRACT
Platelet-activating factor (PAF), a phospholipid autacoid with potent effects throughout the innate immune system, is selectively degraded by two small families of PAF acetylhydrolases (PAF-AHs). These Ca2+-independent phospholipases A2 display remarkable specificity for the length of the sn-2 residue, but this selectivity is lost as the residue gains oxygen functions. Two of the PAF-AHs therefore are specific oxidized phospholipid phospholipases that reduce inflammation, but also remove oxidatively truncated phospholipids that induce apoptosis. The roles of these enzymes are manifold, and their separate and combined functions are now being addressed in model systems and clinical studies.
Subject(s)
1-Alkyl-2-acetylglycerophosphocholine Esterase/metabolism , Animals , Apoptosis , Humans , Inflammation , Platelet Activating Factor/metabolism , Substrate SpecificityABSTRACT
One of the early decisions in what became the Human Genome Project was to recruit families that would serve as a reference set, thereby focusing efforts to create human genetic maps on the same sets of DNA samples. The families recruited from Utah provided the most widely used samples in the Centre d'Etudes du Polymorphisme Humain (CEPH) set, were instrumental in generating human linkage maps, and often serve as the benchmark for establishing allele frequency when a new variant is identified. In addition, the immortalized cell lines created from the peripheral blood cells of these subjects are a broadly used resource and have yielded insights in many areas, from the genetics of gene expression to the regulation of telomeres. More recently, these families were recontacted and underwent extensive, protocol-based evaluation to create a phenotypic database, which will aid in the study of the genetic basis of quantitative traits. As with the earlier efforts, this project involved collaborations among many investigators and has yielded insights into multiple traits.
Subject(s)
Chromosome Mapping , Human Genome Project , Quantitative Trait Loci , Aging/genetics , Chromosome Mapping/history , Chromosomes, Human/genetics , Cooperative Behavior , Female , Gene Expression , History, 20th Century , History, 21st Century , Human Genome Project/history , Humans , Male , Pedigree , Polymorphism, Genetic , Receptors, G-Protein-Coupled/genetics , Telomere/genetics , UtahABSTRACT
The leukocyte integrins have critical roles in host defense and inflammatory tissue injury. We found that integrin alphaDbeta2, a novel but largely uncharacterized member of this family, is restricted to subsets of macrophages and a small population of circulating leukocytes in wild-type mice in the absence of inflammatory challenge and is expressed in regulated fashion during cytokine-induced macrophage differentiation in vitro. alphaDbeta2 is highly displayed on splenic red pulp macrophages and mediates their adhesion to local targets, identifying key functional activity. In response to challenge with Plasmodium berghei, a malarial pathogen that models systemic infection and inflammatory injury, new populations of alphaD+ macrophages evolved in the spleen and liver. Unexpectedly, targeted deletion of alphaD conferred a survival advantage in P. berghei infection over a 30-day observation period. Mechanistic studies demonstrated that the increased survival of alphaD-/- animals at these time points is not attributed to differences in magnitude of anemia or parasitemia or to alterations in splenic microanatomy, each of which is a key variable in the natural history of P. berghei infection, and indicated that an altered pattern of inflammatory cytokines may contribute to the difference in mortality. In contrast to the outcome in malarial challenge, death of alphaD-/- animals was accelerated in a model of Salmonella sepsis, demonstrating differential rather than stereotyped roles for alphaDbeta2 in systemic infection. These studies identify previously unrecognized and unique activities of alphaDbeta2, and macrophages that express it, in host defense and injury.
Subject(s)
CD11 Antigens/metabolism , Integrin alpha Chains/metabolism , Macrophages/immunology , Malaria/immunology , Plasmodium berghei , Salmonella , Sepsis/immunology , Animals , CD11 Antigens/genetics , Cytokines/metabolism , Inflammation/immunology , Integrin alpha Chains/genetics , Liver/immunology , Macrophage Activation , Malaria/mortality , Mice , Mice, Knockout , Sepsis/mortality , Spleen/immunologyABSTRACT
Retinoic acid inducible gene-I (RIG-I) plays important roles during innate immune responses to viral infections and as a transducer of cytokine signaling. The mechanisms of RIG-I up-regulation after cytokine stimulation are incompletely characterized. It was previously reported that IFN-gamma induces the expression of RIG-I in endothelial cells. In this study, we characterized the mechanism of type I IFN-mediated up-regulation of RIG-I in HeLa cells and found that, in addition to type I IFN, TNF-alpha, a cytokine that regulates innate immune responses, induced expression of RIG-I. To investigate whether TNF-alpha- and type I IFN-mediated up-regulations of RIG-I were causally related, we studied the kinetics of these responses. Our results were consistent with a model in which TNF-alpha functioned upstream of type I IFNs. The ability of TNF-alpha to up-regulate RIG-I required protein synthesis, expression of functional type I IFNRs, and STAT1 signaling. We also found that IFN-epsilon was the only IFN isoform expressed constitutively in HeLa cells and that its expression was up-regulated in response to stimulation with TNF-alpha. The mechanism of up-regulation involved stabilization of IFN-epsilon mRNA in the absence of transcriptional activation. Silencing the expression of IFN-epsilon attenuated STAT1 expression and phosphorylation and inhibited RIG-I expression, providing additional support for the participation of IFN-epsilon upstream of STAT1. Our findings support a sequential mechanism whereby TNF-alpha leads to stabilization of IFN-epsilon mRNA, increased IFN-epsilon synthesis, engagement of type I IFNRs, increased STAT1 expression and phosphorylation, and up-regulation of RIG-I expression. These findings have implications for our understanding of the immune responses that follow cytokine stimulation.
Subject(s)
DEAD-box RNA Helicases/metabolism , Interferons/metabolism , STAT1 Transcription Factor/metabolism , Tumor Necrosis Factor-alpha/pharmacology , Uterine Cervical Neoplasms/metabolism , DEAD Box Protein 58 , Female , HeLa Cells , Humans , Interferons/genetics , Phosphorylation/drug effects , Promoter Regions, Genetic/genetics , RNA, Messenger/genetics , Receptors, Immunologic , Receptors, Interferon/classification , Receptors, Interferon/metabolism , Signal Transduction , Up-Regulation/drug effects , Uterine Cervical Neoplasms/geneticsABSTRACT
Mutations in adenomatous polyposis coli (APC) underlie the earliest stages of colorectal carcinogenesis. Consequences of APC mutation include stabilization of beta-catenin, dysregulation of cyclooxygenase-2 (COX-2) expression, and loss of retinoic acid production, events with poorly defined interactions. Here we showed that treatment of zebrafish expressing a truncated form of Apc with either retinoic acid or a selective COX-2 inhibitor decreased beta-catenin protein levels and downstream signaling events. Interestingly, the destruction of beta-catenin in apc mutant embryos following Cox-2 inhibition required the presence of truncated Apc. These findings support roles for retinoic acid and Cox-2 in regulating the stability of beta-catenin following Apc loss. Furthermore, truncated Apc appears to retain the ability to target beta-catenin for destruction, but only in the absence of Cox-2 activity. This novel function of truncated Apc may provide a molecular basis for the efficacy of COX-2 inhibitors in the treatment of colon cancer.
Subject(s)
Adenomatous Polyposis Coli/metabolism , Cyclooxygenase 2/metabolism , Gene Expression Regulation , Mutation , Tretinoin/pharmacology , beta Catenin/metabolism , Animals , Dinoprostone/metabolism , Down-Regulation , Immunoblotting , In Situ Hybridization , RNA/metabolism , Signal Transduction , Zebrafish , beta Catenin/antagonists & inhibitorsABSTRACT
Platelet-activating factor (PAF) is a proinflammatory mediator that plays a central role in acute lung injury (ALI). PAF- acetylhydrolases (PAF-AHs) terminate PAF's signals and regulate inflammation. In this study, we describe the kinetics of plasma and bronchoalveolar lavage (BAL) PAF-AH in the early phase of ALI. Six pigs with oleic acid induced ALI and two healthy controls were studied. Plasma and BAL samples were collected every 2h and immunohistochemical analysis of PAF-AH was performed in lung tissues. PAF-AH activity in BAL was increased at the end of the experiment (BAL PAF-AH Time 0=0.001+/-0.001 nmol/ml/min/g vs Time 6=0.031+/-0.018 nmol/ml/min/g, p=0.04) while plasma activity was not altered. We observed increased PAF-AH staining of macrophages and epithelial cells in the lungs of animals with ALI but not in healthy controls. Our data suggest that increases in PAF-AH levels are, in part, a result of alveolar production. PAF-AH may represent a modulatory strategy to counteract the excessive pro-inflammatory effects of PAF and PAF-like lipids in lung inflammation.
Subject(s)
1-Alkyl-2-acetylglycerophosphocholine Esterase/biosynthesis , Lung/enzymology , Respiratory Distress Syndrome/enzymology , 1-Alkyl-2-acetylglycerophosphocholine Esterase/blood , Animals , Bronchoalveolar Lavage Fluid/chemistry , Female , Immunohistochemistry , Kinetics , Lung/metabolism , Oleic Acid , Respiratory Distress Syndrome/chemically induced , Swine , Time FactorsABSTRACT
Cyclooxygenase-2 is often highly expressed in epithelial malignancies and likely has an active role in tumor development. But how it promotes tumorigenesis is not clearly defined. Recent evidence suggests that this may involve transactivation of the epidermal growth factor receptor through E-prostanoid receptors, but reports differ about the mechanism by which this occurs. We found that E-prostanoid receptors 2-4, but not 1, transactivated the epidermal growth factor receptor. This required metalloproteinase activity, leading to release of growth factors from the cell surface. Both transforming growth factor-alpha and amphiregulin were released in response to over-expression of cyclooxygenase-2, but betacellulin and heparin-binding EGF-like growth factor were not. The metalloproteinase tumor necrosis factor-alpha converting enzyme was required for proteolytic release of transforming growth factor-alpha. We also found that addition of epidermal growth factor receptor ligands to HEK293 cells induced cyclooxygenase-2 expression, suggesting that by activating epidermal growth factor receptor signaling, cyclooxygenase-2 potentially creates a self-perpetuating cycle of cell growth. Consistent with this, inhibition of cyclooxygenase-2 reduced growth of epidermal growth factor receptor over-expressing MCF-10A breast epithelial cells in three-dimensional culture.
Subject(s)
ADAM Proteins/metabolism , Cyclooxygenase 2/metabolism , ErbB Receptors/genetics , Membrane Proteins/metabolism , Receptors, Prostaglandin E/metabolism , Transcriptional Activation/genetics , ADAM17 Protein , Animals , COS Cells , Cell Culture Techniques , Cell Proliferation/drug effects , Chlorocebus aethiops , Cyclooxygenase 2/genetics , Dinoprostone/pharmacology , Enzyme Inhibitors/pharmacology , Epithelial Cells/cytology , Epithelial Cells/drug effects , Epithelial Cells/enzymology , Gene Expression Regulation, Enzymologic/drug effects , Humans , Intercellular Signaling Peptides and Proteins/metabolism , Membrane Proteins/antagonists & inhibitors , Membrane Proteins/genetics , Metalloproteases/metabolism , Mice , RNA, Messenger/genetics , RNA, Messenger/metabolism , Transcriptional Activation/drug effects , Transforming Growth Factor alpha/metabolismABSTRACT
Seven-transmembrane receptor (7TMR) signaling is transduced by second messengers such as diacylglycerol (DAG) generated in response to the heterotrimeric guanine nucleotide-binding protein Gq and is terminated by receptor desensitization and degradation of the second messengers. We show that beta-arrestins coordinate both processes for the Gq-coupled M1 muscarinic receptor. beta-Arrestins physically interact with diacylglycerol kinases (DGKs), enzymes that degrade DAG. Moreover, beta-arrestins are essential for conversion of DAG to phosphatidic acid after agonist stimulation, and this activity requires recruitment of the beta-arrestin-DGK complex to activated 7TMRs. The dual function of beta-arrestins, limiting production of diacylglycerol (by receptor desensitization) while enhancing its rate of degradation, is analogous to their ability to recruit adenosine 3',5'-monophosphate phosphodiesterases to Gs-coupled beta2-adrenergic receptors. Thus, beta-arrestins can serve similar regulatory functions for disparate classes of 7TMRs through structurally dissimilar enzymes that degrade chemically distinct second messengers.
Subject(s)
Arrestins/metabolism , Diacylglycerol Kinase/metabolism , Diglycerides/metabolism , Receptor, Muscarinic M1/metabolism , Signal Transduction , Animals , COS Cells , Carbachol/pharmacology , Cell Line , Chlorocebus aethiops , Diacylglycerol Kinase/genetics , Humans , Mutation , Phosphatidic Acids/metabolism , Protein Binding , RNA, Small Interfering , Recombinant Fusion Proteins/metabolism , Second Messenger Systems , Transfection , beta-ArrestinsABSTRACT
We performed a multitiered, case-control association study of psoriasis in three independent sample sets of white North American individuals (1,446 cases and 1,432 controls) with 25,215 genecentric single-nucleotide polymorphisms (SNPs) and found a highly significant association with an IL12B 3'-untranslated-region SNP (rs3212227), confirming the results of a small Japanese study. This SNP was significant in all three sample sets (odds ratio [OR](common) 0.64, combined P [Pcomb]=7.85x10(-10)). A Monte Carlo simulation to address multiple testing suggests that this association is not a type I error. The coding regions of IL12B were resequenced in 96 individuals with psoriasis, and 30 additional IL12B-region SNPs were genotyped. Haplotypes were estimated, and genotype-conditioned analyses identified a second risk allele (rs6887695) located approximately 60 kb upstream of the IL12B coding region that exhibited association with psoriasis after adjustment for rs3212227. Together, these two SNPs mark a common IL12B risk haplotype (OR(common) 1.40, Pcomb=8.11x10(-9)) and a less frequent protective haplotype (OR(common) 0.58, Pcomb=5.65x10(-12)), which were statistically significant in all three studies. Since IL12B encodes the common IL-12p40 subunit of IL-12 and IL-23, we individually genotyped 17 SNPs in the genes encoding the other chains of these cytokines (IL12A and IL23A) and their receptors (IL12RB1, IL12RB2, and IL23R). Haplotype analyses identified two IL23R missense SNPs that together mark a common psoriasis-associated haplotype in all three studies (OR(common) 1.44, Pcomb=3.13x10(-6)). Individuals homozygous for both the IL12B and the IL23R predisposing haplotypes have an increased risk of disease (OR(common) 1.66, Pcomb=1.33x10(-8)). These data, and the previous observation that administration of an antibody specific for the IL-12p40 subunit to patients with psoriasis is highly efficacious, suggest that these genes play a fundamental role in psoriasis pathogenesis.
Subject(s)
Genetic Predisposition to Disease , Interleukin-12 Subunit p40/genetics , Psoriasis/genetics , Receptors, Interleukin/genetics , Adolescent , Adult , Bayes Theorem , Case-Control Studies , Child , Female , Genetics, Population , Haplotypes , Humans , Male , Middle Aged , Monte Carlo Method , Polymorphism, Single Nucleotide , Receptors, Interleukin-12/geneticsABSTRACT
Platelet-activating factor acetylhydrolase (PAF-AH) is a phospholipase A2 that inactivates potent lipid messengers, such as PAF and modified phospholipids generated in settings of oxidant stress. The catalytic activity of PAF-AH is sensitive to oxidants, a feature that may have pathological consequences. We report that peroxynitrite, an oxidant species generated after cellular activation, mediates oxidative inactivation of PAF-AH. We found that peroxynitrite inactivated and derivatized the recombinant protein and obtained evidence supporting a role for a methionine and two tyrosine residues in this process. We employed interspecies comparisons and site-directed mutagenesis and identified a role for M-117, and a smaller contribution of Y-307 and Y-335 as targets of oxidant attack using free and lipoprotein-associated recombinant proteins. M-117 is adjacent to W-115 and L-116, which are essential for association of PAF-AH with LDL. Oxidation of LDL-associated PAF-AH partially dissociated the enzyme from the particles. Similarly, oxidation of the purified enzyme in the absence of lipoproteins prevented subsequent association with LDL. These results provide new insights into the molecular mechanisms that mediate inactivation of PAF-AH in settings of oxidant stress and the consequences of oxidation on the ability of this enzyme to associate with LDL.
Subject(s)
1-Alkyl-2-acetylglycerophosphocholine Esterase/blood , 1-Alkyl-2-acetylglycerophosphocholine Esterase/chemistry , Animals , COS Cells , Chlorocebus aethiops , Humans , Lipoproteins/chemistry , Mice , Oxidants/metabolism , Oxidative Stress , Oxygen/metabolism , Peroxynitrous Acid/pharmacology , Phospholipases A/metabolism , Phospholipases A2 , Phospholipids/metabolism , Reactive Oxygen Species , Tyrosine/chemistryABSTRACT
The temporal association between loss of function of the tumor suppressor adenomatous polyposis coli (APC) and overexpression of cyclooxygenase 2 (COX-2) has been demonstrated in vivo and has led to the hypothesis that APC regulates COX-2 expression. This could potentially occur through a variety of mechanisms including the well-characterized ability of APC to negatively regulate Wnt signaling and decrease expression of target genes. However, recent findings suggest that the products of COX-2 elicit effects that occur upstream of the beta-catenin/TCF/LEF pathway. This review will focus on the regulation of COX-2 by APC and the interplay between COX-2 and the Wnt signaling pathway.
Subject(s)
Colonic Neoplasms/physiopathology , Cyclooxygenase 2/physiology , Prostaglandins/physiology , Signal Transduction/physiology , Adenomatous Polyposis Coli Protein/physiology , Animals , Dinoprostone/physiology , Gene Expression Regulation, Neoplastic , Genes, APC/physiology , Humans , Up-Regulation , Wnt Proteins/physiology , beta Catenin/physiologyABSTRACT
BACKGROUND: Treatments for recurrent meningiomas are limited. We previously demonstrated universal expression of COX-2 in meningiomas and dose-dependent growth inhibition in vitro with celecoxib, a COX-2 inhibitor. We therefore tested the effects of celecoxib on meningioma growth in a mouse xenograft model. METHODS: Meningioma cell lines (IOMM-Lee, CH157-MN, WHO grade I primary cultured tumor) were transplanted into flanks of nude mice fed mouse chow with celecoxib at varying concentrations (0, 500, 1000, 1500 ppm) ad libitum. Tumors were measured biweekly and processed for MIB-1, Factor VIII, COX-2, and VEGF, and assayed with transferase-mediated dUTP-biotin nick-end labeling (TUNEL). RESULTS: Celecoxib reduced growth of mean tumor volume by 66% (P < .05), 25% (P > .05), and 65% (P < .05) compared with untreated controls in IOMM-Lee, CH157-MN, and benign tumors, respectively. IOMM-Lee tumors removed from celecoxib treatment regained a growth rate similar to the control. Blood vessel density decreased and apoptotic cells increased in treated flank tumors. Diminished COX-2 expression and VEGF were observed in treated IOMM-Lee tumors. Mean plasma celecoxib levels were 845, 1540, and 2869 ng/mL, for low-, medium-, and high-dose celecoxib, respectively. CONCLUSIONS: Celecoxib inhibits meningioma growth in vivo at plasma levels achievable in humans. Celecoxib-treated tumors were less vascular with increased apoptosis. IOMM-Lee tumors treated with celecoxib showed decreased COX-2 and VEGF expression. COX-2 inhibitors may have a role in the treatment of recurrent meningiomas.
Subject(s)
Cyclooxygenase Inhibitors/therapeutic use , Meningeal Neoplasms/prevention & control , Meningioma/prevention & control , Pyrazoles/therapeutic use , Sulfonamides/therapeutic use , Animals , Apoptosis , Celecoxib , Cyclooxygenase 2/metabolism , Factor VIII/metabolism , Humans , Immunoenzyme Techniques , In Situ Nick-End Labeling , Ki-67 Antigen/metabolism , Meningeal Neoplasms/enzymology , Meningeal Neoplasms/pathology , Meningioma/enzymology , Meningioma/pathology , Mice , Mice, Nude , Survival Rate , Tumor Cells, Cultured , Vascular Endothelial Growth Factor A/metabolism , Xenograft Model Antitumor AssaysABSTRACT
Diacylglycerol kinases (DGKs) phosphorylate diacylglycerol (DAG) to terminate its signaling. To study DGKdelta, we disrupted its gene in mice and found that DGKdelta deficiency reduced EGF receptor (EGFR) protein expression and activity. Similar to EGFR knockout mice, DGKdelta-deficient pups were born with open eyelids and died shortly after birth. PKCs are activated by DAG and phosphorylate EGFR to reduce its expression and activity. We found DAG accumulation, increased threonine phosphorylation of EGFR, enhanced phosphorylation of other PKC substrates, and increased PKC autophosphorylation in DGKdelta knockout cells, indicating that DGKdelta regulates EGFR by modulating PKC signaling.
Subject(s)
Diacylglycerol Kinase/metabolism , ErbB Receptors/metabolism , Isoenzymes/metabolism , Protein Kinase C/metabolism , Signal Transduction/physiology , Animals , Cells, Cultured , Diacylglycerol Kinase/genetics , Diglycerides/metabolism , Enzyme Activation , ErbB Receptors/genetics , Female , Gene Deletion , Humans , Intercellular Signaling Peptides and Proteins/genetics , Intercellular Signaling Peptides and Proteins/metabolism , Isoenzymes/genetics , JNK Mitogen-Activated Protein Kinases/metabolism , Keratinocytes/cytology , Keratinocytes/physiology , Mice , Mice, Inbred C57BL , Mice, Knockout , Phenotype , Phosphorylation , Protein Kinase C/genetics , Threonine/metabolismABSTRACT
Tight regulation of COX-2 expression is a key feature controlling eicosanoid production in atherosclerosis and other inflammatory syndromes. Adhesive interactions between platelets and monocytes occur in these conditions and deliver specific signals that trigger inflammatory gene expression. Using a cellular model of monocyte signaling induced by activated human platelets, we identified the central posttranscriptional mechanisms that regulate timing and magnitude of COX-2 expression. Tethering of monocytes to platelets and to purified P-selectin, a key adhesion molecule displayed by activated platelets, induces NF-kappaB activation and COX-2 promoter activity. Nevertheless, COX-2 mRNA is rapidly degraded, leading to aborted protein synthesis. Time-dependent signaling of monocytes induces a second phase of transcript accumulation accompanied by COX-2 enzyme synthesis and eicosanoid production. Here, generation of IL-1beta, a proinflammatory cytokine, promoted stabilization of COX-2 mRNA by silencing of the AU-rich mRNA decay element (ARE) in the 3'-untranslated region (3'UTR) of the mRNA. Consistent with observed mRNA stabilization, activated platelets or IL-1beta treatment induced cytoplasmic accumulation and enhanced ARE binding of the mRNA stability factor HuR in monocytes. These findings demonstrate that activated platelets induce COX-2 synthesis in monocytes by combinatorial signaling to transcriptional and posttranscriptional checkpoints. These checkpoints may be altered in disease and therefore useful as targets for antiinflammatory intervention.
Subject(s)
Blood Platelets/metabolism , Cell Communication/physiology , Cyclooxygenase 2/genetics , Cytokines/metabolism , Membrane Proteins/genetics , Monocytes/metabolism , Signal Transduction/physiology , 3' Untranslated Regions/genetics , Active Transport, Cell Nucleus/physiology , Antigens, Surface/metabolism , Blood Platelets/cytology , Cell Adhesion/genetics , Cell Adhesion/physiology , Cell Communication/genetics , Cytokines/pharmacology , Dinoprostone/metabolism , ELAV Proteins , ELAV-Like Protein 1 , Extracellular Signal-Regulated MAP Kinases/metabolism , Gene Expression Regulation, Enzymologic/genetics , Humans , Interleukin 1 Receptor Antagonist Protein/pharmacology , Interleukin-1beta/metabolism , Interleukin-1beta/pharmacology , Monocytes/cytology , NF-kappa B/metabolism , P-Selectin/pharmacology , Platelet Activation/physiology , Poly(A)-Binding Proteins/metabolism , RNA Stability/drug effects , RNA-Binding Proteins/metabolism , T-Cell Intracellular Antigen-1 , Thrombin/pharmacology , Transfection , U937 Cells , p38 Mitogen-Activated Protein Kinases/metabolismABSTRACT
Current evidence indicates that dysregulation of the host inflammatory response to infectious agents is central to the mortality of patients with sepsis and in those with systemic inflammatory response syndrome. Strategies to block inflammatory mediators, often with complicated outcomes, are currently being investigated as new adjuvant therapies for sepsis. Here, we determined if administration of recombinant platelet-activating factor (rPAF)-acetylhydrolase (rPAF-AH), an enzyme that inactivates PAF and PAF-like lipids, protects mice from inflammatory injury and death after administration of lipopolysaccharide (LPS) or cecal ligation and puncture (CLP). Administration of rPAF-AH increased plasma PAF-AH activity and reduced mortality in both models. Treatment with rPAF-AH increased peritoneal fluid levels of monocyte chemoattractant protein 1/CCL-2 and decreased interleukin 6 and migration inhibitory factor levels after LPS administration or CLP. Administration of a broad-spectrum antibiotic together with rPAF-AH was more protective than single treatment with either of these agents. The combined treatment was associated with reduced interleukin 6 levels in mice subjected to CLP. We observed acute decreases in plasma PAF-AH activity in mice subjected to CLP or challenged with LPS and in human patients with sepsis. We conclude that alterations in the endogenous PAF-AH contribute to the pathophysiology of sepsis and that administration of exogenous rPAF-AH reduces inflammatory injury and mortality in models relevant to the clinical syndrome. Variations in endogenous PAF-AH activity may potentially account for variable responses to exogenous rPAF-AH in previous clinical trials. Serial measurements of plasma PAF-AH activity in murine models demonstrate dynamic regulation of the endogenous enzyme, potentially explaining the variations in human subjects.