RESUMEN
Essentials Platelets express retinoic acid receptor (RAR)α protein, specifically binding target mRNAs. mRNAs under RARα control include MAP1LC3B2, SLAIN2, and ANGPT1. All-trans retinoic acid (atRA) releases RARα from its target mRNA. RARα expressed in human platelets exerts translational control via direct mRNA binding. SUMMARY: Background Translational control mechanisms in platelets are incompletely defined. Here, we determined whether the nuclear transcription factor RARα controls protein translational events in human platelets. Methods Isolated human platelets were treated with the pan-RAR agonist all-trans-retinoic acid (atRA). Global and targeted translational events were examined. Results Stimulation of platelets with atRA significantly increased global protein expression. RARα protein bound to a subset of platelet mRNAs, as measured by next-generation RNA-sequencing. In-depth analyses of 5' and 3'-untranslated regions of the RARα-bound mRNAs revealed consensus RARα binding sites in microtubule-associated protein 1 light chain 3 beta 2 (MAP1LC3B2), SLAIN motif-containing protein 2 (SLAIN2) and angiopoietin-1 (ANGPT1) transcripts. When platelets were treated with atRA, binding interactions between RARα protein and mRNA for MAP1LC3B2, SLAIN2 and ANGPT1 were significantly decreased. Consistent with the release of bound RARα protein from MAP1LCB2mRNA, we observed an increase in the synthesis of MAP1LC3B2 protein. Conclusions These findings provide the first evidence that RARα, a nuclear transcriptional factor, regulates synthetic events in anucleate human platelets. They also reveal an additional non-genomic role for RARα in platelets that may have implications for the vitamin A-dependent signaling in humans.
Asunto(s)
Plaquetas/metabolismo , Proteínas Sanguíneas/biosíntesis , Proteínas Sanguíneas/genética , Receptor alfa de Ácido Retinoico/sangre , Angiopoyetina 1/biosíntesis , Angiopoyetina 1/sangre , Angiopoyetina 1/genética , Secuencia de Bases , Sitios de Unión/genética , Plaquetas/efectos de los fármacos , Humanos , Técnicas In Vitro , Proteínas Asociadas a Microtúbulos/biosíntesis , Proteínas Asociadas a Microtúbulos/sangre , Proteínas Asociadas a Microtúbulos/genética , Biosíntesis de Proteínas , ARN Mensajero/sangre , ARN Mensajero/genética , Receptor alfa de Ácido Retinoico/genética , Tretinoina/farmacologíaRESUMEN
Essentials Co-existent damaging variants are likely to cause more severe bleeding and may go undiagnosed. We determined pathogenic variants in a three-generational pedigree with excessive bleeding. Bleeding occurred with concurrent variants in prostaglandin synthase-1 (PTGS-1) and factor VIII. The PTGS-1 variant was associated with functional defects in the arachidonic acid pathway. SUMMARY: Background Inherited human variants that concurrently cause disorders of primary hemostasis and coagulation are uncommon. Nevertheless, rare cases of co-existent damaging variants are likely to cause more severe bleeding and may go undiagnosed. Objective We prospectively sought to determine pathogenic variants in a three-generational pedigree with excessive bleeding. Patients/methods Platelet number, size and light transmission aggregometry to multiple agonists were evaluated in pedigree members. Transmission electron microscopy determined platelet morphology and granule content. Thromboxane release studies and light transmission aggregometry in the presence or absence of prostaglandin G2 assessed specific functional defects in the arachidonic acid pathway. Whole exome sequencing (WES) and targeted nucleotide sequence analysis identified potentially deleterious variants. Results Pedigree members with excessive bleeding had impaired platelet aggregation with arachidonic acid, epinephrine and low-dose ADP, as well as reduced platelet thromboxane B2 release. Impaired platelet aggregation in response to 2MesADP was rescued with prostaglandin G2 , a prostaglandin intermediate downstream of prostaglandin synthase-1 (PTGS-1) that aids in the production of thromboxane. WES identified a non-synonymous variant in the signal peptide of PTGS-1 (rs3842787; c.50C>T; p.Pro17Leu) that completely co-segregated with disease phenotype. A variant in the F8 gene causing hemophilia A (rs28935203; c.5096A>T; p.Y1699F) was also identified. Individuals with both variants had more severe bleeding manifestations than characteristic of mild hemophilia A alone. Conclusion We provide the first report of co-existing variants in both F8 and PTGS-1 genes in a three-generation pedigree. The PTGS-1 variant was associated with specific functional defects in the arachidonic acid pathway and more severe hemorrhage.
Asunto(s)
Factor VIII/genética , Hemorragia/genética , Prostaglandina-Endoperóxido Sintasas/genética , Adulto , Anciano , Ácido Araquidónico/metabolismo , Niño , Ciclooxigenasa 1/genética , Salud de la Familia , Femenino , Frecuencia de los Genes , Variación Genética , Hemorragia/sangre , Hemorragia/inmunología , Humanos , Masculino , Persona de Mediana Edad , Linaje , Agregación Plaquetaria , Recuento de Plaquetas , Estudios Prospectivos , Prostaglandina-Endoperóxido Sintasas/sangre , Tromboxano B2/genética , Adulto JovenRESUMEN
UNLABELLED: Essentials Platelets employ proteins/signaling pathways traditionally thought reserved for nuclear niche. We determined retinoic-acid-receptor alpha (RARα) expression and function in human platelets. RARα/actin-related protein-2/3 complex (Arp2/3) interact via non-genomic signaling in platelets. RARα regulates Arp2/3-mediated actin cytoskeletal dynamics and platelet spreading. SUMMARY: Background Platelets utilize proteins and pathways classically reserved for the nuclear niche. Methods We determined whether human platelets express retinoic-acid-receptor family members, traditionally thought of as nuclear transcription factors, and deciphered the function of RARα. Results We found that RARα is robustly expressed in human platelets and megakaryocytes and interacts directly with actin-related protein-2/3 complex (Arp2/3) subunit 5 (Arp2/3s5). Arp2/3s5 co-localized with RARα in situ and regulated platelet cytoskeletal processes. The RARα ligand all-trans retinoic acid (atRA) disrupted RARα-Arp2/3 interactions. When isolated human platelets were treated with atRA, rapid cytoskeletal events (e.g. platelet spreading) were inhibited. In addition, when platelets were cultured for 18 h in the presence of atRA, actin-dependent morphological changes (e.g. extended cell body formation) were similarly inhibited. Using in vitro actin branching assays, RARα and Arp2/3-regulated complex actin branch formation was demonstrated. Consistent with inhibition of cytoskeletal processes in platelets, atRA, when added to this branching assay, resulted in dysregulated actin branching. Conclusion Our findings identify a previously unknown mechanism by which RARα regulates Arp2/3-mediated actin cytoskeletal dynamics through a non-genomic signaling pathway. These findings have broad implications in both nucleated and anucleate cells, where actin cytoskeletal events regulate cell morphology, movement and division.
Asunto(s)
Actinas/metabolismo , Plaquetas/metabolismo , Citoesqueleto/metabolismo , Receptor alfa de Ácido Retinoico/metabolismo , Proteína 2 Relacionada con la Actina/metabolismo , Proteína 3 Relacionada con la Actina/metabolismo , Antígenos CD34/metabolismo , Apoptosis , Perfilación de la Expresión Génica , Voluntarios Sanos , Humanos , Espectrometría de Masas , Microscopía Fluorescente , Transducción de Señal/efectos de los fármacos , Factores de Transcripción/metabolismoRESUMEN
BACKGROUND: Worldwide, dengue is the most prevalent human arbovirus disease. Dengue infection may cause a range of clinical manifestations from self-limiting febrile illness through to a life-threatening syndrome accompanied by both bleeding and shock. Thrombocytopenia is frequently observed in mild and severe disease; however, the mechanisms involved in DENV-induced platelet activation and thrombocytopenia are incompletely understood. PATIENTS AND METHODS: Freshly isolated platelets from patients with dengue were evaluated for markers of activation, mitochondrial alteration and activation of cell death pathways. In parallel, we examined direct DENV-induced activation and apoptosis of platelets obtained from healthy subjects. RESULTS: We found that platelets from DENV-infected patients exhibited increased activation by comparison to control subjects. Moreover, platelets from DENV-infected patients exhibited classic signs of the intrinsic pathway of apoptosis that include increased surface phosphatidylserine exposure, mitochondrial depolarization and activation of caspase-9 and -3. Indeed, thrombocytopenia was shown to strongly associate with enhanced platelet activation and cell death in DENV-infected patients. Platelet activation, mitochondrial dysfunction and caspase-dependent phosphatidylserine exposure on platelets were also observed when platelets from healthy subjects were directly exposed to DENV in vitro. DENV-induced platelet activation was shown to occur through mechanisms largely dependent on DC-SIGN. CONCLUSIONS: Together our results demonstrate that platelets from patients with dengue present signs of activation, mitochondrial dysfunction and activation of the apoptosis caspase cascade, which may contribute to the development of thrombocytopenia in patients with dengue. Our results also suggest the involvement of DC-SIGN as a critical receptor in DENV-dependent platelet activation.
Asunto(s)
Caspasas/fisiología , Moléculas de Adhesión Celular/fisiología , Muerte Celular/fisiología , Virus del Dengue/fisiología , Lectinas Tipo C/fisiología , Mitocondrias/fisiología , Activación Plaquetaria/fisiología , Receptores de Superficie Celular/fisiología , Adulto , Estudios de Casos y Controles , Femenino , Humanos , Masculino , Persona de Mediana EdadRESUMEN
BACKGROUND: Activated platelets have previously-unrecognized mechanisms of post-transcriptional gene expression that may influence hemostasis and inflammation. A novel pathway involves splicing of pre-mRNAs in resting platelets to mature, translatable mRNAs in response to cellular activation. OBJECTIVES: We asked if bacterial products and host agonists present in the septic milieu induce tissue factor pre-mRNA splicing in platelets from healthy subjects. In parallel, we asked if spliced tissue factor (TF) mRNA is present in platelets from septic patients in a proof-of-principle analysis. PATIENTS/METHODS: TF pre-mRNA and mRNA expression patterns were characterized in platelets from septic patients and in platelets isolated from healthy subjects activated with bacteria, toxins and inflammatory agonists. Procoagulant activity was also measured. RESULTS AND CONCLUSIONS: Live bacteria, staphylococcal α-toxin and lipopolysaccharide (LPS) induced TF pre-mRNA splicing in platelets isolated from healthy subjects. Toxin-stimulated platelets accelerated plasma clotting, a response that was blocked by a previously-characterized splicing inhibitor and by an anti-tissue factor antibody. Platelets from septic patients expressed spliced TF mRNA, whereas it was absent from unselected and age-matched control subjects. Tissue factor-dependent procoagulant activity was elevated in platelets from a subset of septic patients. Thus, bacterial and host factors induce splicing of TF pre-mRNA, expression of TF mRNA and tissue factor-dependent clotting activity in human platelets. TF mRNA is present in platelets from some septic patients, indicating that it may be a marker of altered platelet phenotype and function in sepsis and that splicing pathways are induced in this syndrome.
Asunto(s)
Plaquetas/metabolismo , Empalme del ARN , ARN Mensajero/metabolismo , Sepsis/metabolismo , Secuencia de Bases , Cartilla de ADN , HumanosRESUMEN
In the late 1960s, numerous investigators independently demonstrated that platelets are capable of synthesizing proteins. Studies continued at a steady pace over the next 30 years and into the 21st century. Collectively, these investigations confirmed that platelets synthesize proteins and that the pattern of protein synthesis changes in response to cellular activation. More recent studies have characterized the mechanisms by which platelets synthesize proteins and have shown that protein synthesis alters the phenotype and functions of platelets. Here, we chronologically review our increased understanding of protein synthetic responses in platelets and discuss how the field may evolve over the next decade.
Asunto(s)
Plaquetas/metabolismo , Biosíntesis de Proteínas , Animales , Investigación Biomédica/historia , Investigación Biomédica/tendencias , Historia del Siglo XX , Historia del Siglo XXI , Humanos , Activación PlaquetariaRESUMEN
OBJECTIVE: To compare the pattern of lung uptake of 18F-fluorodeoxyglucose (FDG) by positron emission tomography (PET) imaging in patients with lung contusion that developed or did not progress to acute respiratory distress syndrome (ARDS). DESIGN: Prospective, observational study. SETTING: Trauma Center (academic urban hospital). PATIENTS AND INTERVENTIONS: Eight patients with blunt thoracic trauma and pulmonary contusion, confirmed by computed tomography (CT) on admission, underwent repeat CT and FDG-PET (on the same day) 24-72 h after admission. RESULTS: No subjects met the criteria for ARDS at the time of the PET and second CT. Four subjects subsequently developed ARDS 1-3 days after the PET scan; the other four did not develop the syndrome. Three of the four subjects who subsequently developed ARDS showed diffuse FDG uptake throughout the entire lungs, while those who did not develop ARDS showed significant FDG uptake only in areas of focal lung opacity (non or poorly aerated lung units) on CT. FDG uptake in normally aerated lung regions was higher for those who subsequently developed ARDS than those who did not, approaching statistical significance. The normally aerated tissue:liver ratio was significantly higher in subjects who developed ARDS than in those who did not (P = 0.029). CONCLUSION: In this small series of patients with thoracic trauma, diffuse lung uptake of FDG was detected by PET imaging 1-3 days prior to clinically determined ARDS.
Asunto(s)
Tomografía de Emisión de Positrones , Síndrome de Dificultad Respiratoria/diagnóstico , Lesión Pulmonar Aguda/complicaciones , Adolescente , Adulto , Anciano , Estudios de Cohortes , Diagnóstico Precoz , Femenino , Fluorodesoxiglucosa F18 , Humanos , Masculino , Persona de Mediana Edad , Proyectos Piloto , Valor Predictivo de las Pruebas , Radiofármacos , Síndrome de Dificultad Respiratoria/etiología , Centros Traumatológicos , Adulto JovenAsunto(s)
Tromboembolia/clasificación , Trombosis de la Vena/clasificación , Animales , Arteriosclerosis/sangre , Coagulación Sanguínea/fisiología , Humanos , Inflamación/sangre , Leucocitos/fisiología , Factores de Riesgo , Tromboembolia/sangre , Tromboembolia/diagnóstico , Trombosis de la Vena/sangre , Trombosis de la Vena/diagnósticoRESUMEN
Platelets are small in size and simple in structure. Nevertheless, these anucleate cytoplasts utilize complex molecular systems to regulate a variety of biological functions. Here we review evolutionary paths, traditional roles, and previously unrecognized biological capacities of platelets that interface thrombosis with inflammation and potentially identify new roles in inflammatory diseases.
Asunto(s)
Plaquetas/fisiología , Inflamación/etiología , Plaquetas/metabolismo , Humanos , Inflamación/sangre , Integrinas/fisiología , Filogenia , Activación Plaquetaria , Trombosis/sangre , Trombosis/etiologíaRESUMEN
Cyclooxygenase-2 (COX-2) expression is normally tightly regulated. However, constitutive overexpression plays a key role in colon carcinogenesis. To understand the molecular nature of enhanced COX-2 expression detected in colon cancer, we examined the ability of the AU-rich element-containing (ARE-containing) 3' untranslated region (3'UTR) of COX-2 mRNA to regulate rapid mRNA decay in human colon cancer cells. In tumor cells displaying enhanced growth and tumorigenicity that is correlated with elevated COX-2, vascular endothelial growth factor (VEGF), and IL-8 protein levels, the corresponding mRNAs were transcribed constitutively and turned over slowly. The observed mRNA stabilization is owing to defective recognition of class II-type AREs present within the COX-2, VEGF, and IL-8 3'UTRs; c-myc mRNA, containing a class I ARE decayed rapidly in the same cells. Correlating with cellular defects in mRNA stability, the RNA-binding of trans-acting cellular factors was altered. In particular, we found that the RNA-stability factor HuR binds to the COX-2 ARE, and overexpression of HuR, as detected in tumors, results in elevated expression of COX-2, VEGF, and IL-8. These findings demonstrate the functional significance rapid mRNA decay plays in controlling gene expression and show that dysregulation of these trans-acting factors can lead to overexpression of COX-2 and other angiogenic proteins, as detected in neoplasia.
Asunto(s)
Antígenos de Superficie , Neoplasias del Colon/enzimología , Isoenzimas/genética , Prostaglandina-Endoperóxido Sintasas/genética , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/fisiología , Regiones no Traducidas 3'/metabolismo , Ciclooxigenasa 2 , Proteínas ELAV , Proteína 1 Similar a ELAV , Factores de Crecimiento Endotelial/genética , Células HT29 , Humanos , Interleucina-8/genética , Linfocinas/genética , Proteínas de la Membrana , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Fosforilación , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial VascularRESUMEN
Atherosclerosis has an underlying inflammatory component. Oxidation of low-density lipoprotein (LDL) particles to modified forms promotes atherogenesis by supplying cholesterol and through the oxidative generation of agents that activate macrophages, smooth muscle and endothelial cells. A primary target of oxidizing compounds, derived from cigarette smoke, dietary sources, exuberant inflammatory cell responses and normal cellular metabolism among other sources, are the esterified polyunsaturated fatty acids in the phospholipid shell that surrounds the insoluble lipids of the lipoprotein core. One type of phospholipid oxidation product mimics the structure of the potent inflammatory mediator platelet-activating factor (PAF), and these oxidation products activate the PAF receptor found on platelets, monocytes and leukocytes. Production of such PAF mimetics is, in contrast to the physiologic generation of PAF, uncontrolled. PAF mimetics and other phospholipid oxidation products are found in atherosclerotic lesions or even in blood after exposure to cigarette smoke. Here we summarize our data describing the structure, activity and metabolism of the PAF-like lipids found in atherogenic LDL particles.
Asunto(s)
Mediadores de Inflamación/metabolismo , Lipoproteínas LDL/metabolismo , Fosfolípidos/metabolismo , Factor de Activación Plaquetaria/metabolismo , Animales , Humanos , Oxidación-ReducciónRESUMEN
All bacteria contain proteins in which their amino-terminal cysteine residue is modified with N-acyl S-diacylglycerol functions, and peptides and proteins bearing this modification are immunomodulatory. The major outer membrane lipoprotein of Escherichia coli, the Braun lipoprotein (BLP), is the prototypical triacylated cysteinyl-modified protein. We find it is as active as LPS in stimulating human endothelial cells to an inflammatory phenotype, and a BLP-negative mutant of E. coli was less inflammatory than its parental strain. While the lipid modification was essential, the lipidated protein was more potent than a lipid-modified peptide. BLP associates with CD14, but this interaction, unlike that with LPS, was not required to elicit endothelial cell activation. BLP stimulated endothelial cell E-selectin surface expression, IL-6 secretion, and up-regulation of the same battery of cytokine mRNAs induced by LPS. Quantitative microarray analysis of 4400 genes showed the same 30 genes were induced by BLP and LPS, and that there was near complete concordance in the level of gene induction. We conclude that the lipid modification of at least one abundant Gram-negative protein is essential for endotoxic activity, but that the protein component also influences activity. The equivalent potency of BLP and LPS, and their complete concordance in the nature and extent of endothelial cell activation show that E. coli endotoxic activity is not due to just LPS. The major outer membrane protein of E. coli is a fully active endotoxic agonist for endothelial cells.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/toxicidad , Endotelio Vascular/efectos de los fármacos , Lipopolisacáridos/toxicidad , Lipoproteínas/toxicidad , Secuencia de Aminoácidos , Adhesión Celular/efectos de los fármacos , Endotelio Vascular/citología , Escherichia coli/patogenicidad , Leucocitos/fisiología , Receptores de Lipopolisacáridos/fisiología , Datos de Secuencia MolecularRESUMEN
Circulating monocytes adhere to platelets and matrix proteins at sites of vascular injury, where engagement of specific surface tethering molecules mediates outside-in signaling and synthesis of gene products by the leukocytes. Here we demonstrate that interaction of isolated human monocytes with collagen induces matrix metalloproteinase-9 (MMP-9; gelatinase B) synthesis by monocytes, a process that is greatly enhanced in the presence of platelets. MMP-9 is a potent matrix degrading enzyme implicated in atherosclerotic plaque rupture, aneurysm formation, and other vascular syndromes. Synthesis of MMP-9 by monocytes is tightly regulated and synergistically increased following adhesion to collagen and platelets. Adhesion to control matrix proteins alone did not result in MMP-9 protein production and, similarly, adhesion of monocytes to platelets activated with thrombin in suspension was not sufficient to induce MMP-9 synthesis in the absence of monocyte adhesion to collagen. Interruption of intercellular contact between platelets and monocytes dramatically inhibited MMP-9 synthesis. These observations demonstrate that discrete adhesion-dependent signaling pathways govern MMP-9 synthesis by monocytes. The synthesis of MMP-9 by monocytes may be critical in vascular syndromes and other pathological processes that are dependent on dysregulated cell-cell and cell-matrix interactions.
Asunto(s)
Plaquetas/citología , Colágeno/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Monocitos/citología , Plaquetas/efectos de los fármacos , Plaquetas/metabolismo , Western Blotting , Adhesión Celular/fisiología , Quimiocina CCL2/genética , Quimiocina CCL2/metabolismo , Técnicas de Cocultivo , Colágeno/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Laminina/metabolismo , Laminina/farmacología , Metaloproteinasa 9 de la Matriz/efectos de los fármacos , Metaloproteinasa 9 de la Matriz/genética , Monocitos/efectos de los fármacos , Monocitos/metabolismo , Unión Proteica , ARN Mensajero/efectos de los fármacos , ARN Mensajero/genética , ARN Mensajero/metabolismoAsunto(s)
Glicoproteínas de Membrana/fisiología , Selectina-P/fisiología , Comunicación Celular/fisiología , Movimiento Celular/fisiología , Dimerización , Humanos , Inflamación/etiología , Leucocitos/fisiología , Ligandos , Glicoproteínas de Membrana/química , Selectina-P/química , Unión Proteica , Estructura Cuaternaria de ProteínaRESUMEN
Platelets release preformed mediators and generate eicosanoids that regulate acute hemostasis and inflammation, but these anucleate cytoplasts are not thought to synthesize proteins or cytokines, or to influence inflammatory responses over time. Interrogation of an arrayed cDNA library demonstrated that quiescent platelets contain many messenger RNAs, one of which codes for interleukin 1beta precursor (pro-IL-1beta). Unexpectedly, the mRNA for IL-1beta and many other transcripts are constitutively present in polysomes, providing a mechanism for rapid synthesis. Platelet activation induces rapid and sustained synthesis of pro-IL-1beta protein, a response that is abolished by translational inhibitors. A portion of the IL-1beta is shed in its mature form in membrane microvesicles, and induces adhesiveness of human endothelial cells for neutrophils. Signal-dependent synthesis of an active cytokine over several hours indicates that platelets may have previously unrecognized roles in inflammation and vascular injury. Inhibition of beta3 integrin engagement markedly attenuated the synthesis of IL-1beta, identifying a new link between the coagulation and inflammatory cascades, and suggesting that antithrombotic therapies may also have novel antiinflammatory effects.
Asunto(s)
Interleucina-1/genética , Interleucina-1/inmunología , Activación Plaquetaria/inmunología , Transducción de Señal/inmunología , Antígenos CD/fisiología , Coagulación Sanguínea/inmunología , Adhesión Celular/inmunología , Endotelio Vascular/citología , Endotelio Vascular/inmunología , Fibrina/fisiología , Expresión Génica/inmunología , Humanos , Integrina beta3 , Neutrófilos/citología , Neutrófilos/inmunología , Glicoproteínas de Membrana Plaquetaria/fisiología , Polirribosomas/genética , Biosíntesis de Proteínas/inmunología , ARN Mensajero/análisisRESUMEN
Lysophosphatidylcholine is an abundant component of plasma and oxidized LDL that displays several biological activities, some of which may occur through the platelet-activating factor (PAF) receptor. We find that commercial lysophosphatidylcholine, its alkyl homolog (lyso-PAF), and PAF all induce inflammation in a murine model of pleurisy. Hydrolysis of PAF to lyso-PAF by recombinant PAF acetylhydrolase abolished this eosinophilic infiltration, implying that lyso-PAF should not have displayed inflammatory activity. Saponification of lyso-PAF or PAF acetylhydrolase treatment of lyso-PAF or lysophosphatidylcholine abolished activity; neither lysolipid should contain susceptible sn-2 residues, suggesting contaminants account for the bioactivity. Lyso-PAF and to a lesser extent lysophosphatidylcholine stimulated Ca(2+) accumulation in 293 cells stably transfected with the human PAF receptor, and this was inhibited by specific PAF receptor antagonists. Again, treatment of lyso-PAF or lysophosphatidylcholine with recombinant PAF acetylhydrolase, a nonselective phospholipase A(2), or saponification of lyso-PAF destroyed the PAF-like activity, a result incompatible with lyso-PAF or lysophosphatidylcholine being the actual agonist. We conclude that neither lyso-PAF nor lysophosphatidylcholine is a PAF receptor agonist, nor are they inflammatory by themselves. We suggest that PAF or a PAF-like mimetic accounts for inflammatory effects of lysophosphatidylcholine and lyso-PAF.
Asunto(s)
Contaminación de Medicamentos , Inflamación/inducido químicamente , Lisofosfatidilcolinas/farmacología , Fosfolípidos/farmacología , Factor de Activación Plaquetaria/análogos & derivados , Factor de Activación Plaquetaria/farmacología , Receptores de Superficie Celular , Receptores Acoplados a Proteínas G , 1-Alquil-2-acetilglicerofosfocolina Esterasa , Animales , Calcio/metabolismo , Fluorescencia , Humanos , Hidrólisis , Lisofosfatidilcolinas/química , Lisofosfatidilcolinas/metabolismo , Ratones , Fosfolipasas A/metabolismo , Factor de Activación Plaquetaria/química , Factor de Activación Plaquetaria/metabolismo , Glicoproteínas de Membrana Plaquetaria/efectos de los fármacos , Glicoproteínas de Membrana Plaquetaria/genética , Glicoproteínas de Membrana Plaquetaria/fisiología , Pleuresia/inducido químicamente , Proteínas Recombinantes/metabolismo , TransfecciónRESUMEN
Engagement of adhesion molecules on monocytes and other myeloid leukocytes, which are effector cells of the innate immune system, not only tethers the leukocytes in place but also transmits outside-in signals that induce functional changes and alter gene expression. We found that a subset of mRNAs that are induced or amplified by adhesion of human monocytes to P-selectin via its surface ligand, P-selectin glycoprotein 1, have characteristics that suggest specialized translational control. One of these codes for urokinase plasminogen activator receptor (UPAR), a critical surface protease receptor and regulator of cell adhesion and migration. Although UPAR transcripts are induced by adhesion, rapid synthesis of the protein uses constitutive mRNA without a requirement for new transcription and is regulated by mammalian target of rapamycin, demonstrating new biologic roles for the signal-dependent translation pathway controlled by this intracellular kinase. The synthesis of UPAR in monocytic cells is also regulated by eukaryotic translation initiation factor 4E, a second key translational checkpoint, and phosphorylation of eukaryotic translation initiation factor 4E is induced by adhesion of monocytes to P-selectin. Translationally controlled display of UPAR by monocytes confers recognition of the matrix protein, vitronectin. Adhesion-dependent signaling from the plasma membrane to translational checkpoints represents a previously unrecognized mechanism for regulating surface phenotype that may be particularly important for myeloid leukocytes and other cells that are specialized for rapid inflammatory and vascular responses.
Asunto(s)
Adhesión Celular/genética , Regulación de la Expresión Génica , Monocitos/fisiología , Membrana Celular/fisiología , Factor 4E Eucariótico de Iniciación , Humanos , Selectina-P/fisiología , Factores de Iniciación de Péptidos/metabolismo , Fenotipo , Fosforilación , Biosíntesis de Proteínas , Proteínas Quinasas/fisiología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores de Superficie Celular/biosíntesis , Receptores del Activador de Plasminógeno Tipo Uroquinasa , Transducción de Señal , Serina-Treonina Quinasas TORRESUMEN
Unmitigated oxidative stress is deleterious, as epitomized by CCl4 intoxication. In this well-characterized model of free radical-initiated damage, liver metabolism of CCl4 to CCl3. causes lipid peroxidation, F-ring isoprostane formation, and pathologic leukocyte activation. The nature of the mediator that couples oxidation to the hepatotoxic inflammatory response is uncharacterized. We found that oxidatively modified phosphatidylcholines were present in the livers of CCl4-exposed rats and not in livers from control animals, that CCl4 metabolism generated lipids that activated 293 cells stably transfected with the human platelet-activating factor (PAF) receptor, and that this PAF-like activity was formed as rapidly as isoprostane-containing phosphatidylcholine (iPC) during oxidation. iPC and the PAF-like activity also had similar chromatographic properties. The potential for iPC activation of the PAF receptor has been unexplored, but we conclude that iPC themselves did not activate the PAF receptor, as phospholipase A1 hydrolysis completely destroyed iPC, but none of the PAF-like bioactivity. Oxidatively fragmented phospholipids are potent agonists of the PAF receptor, but mass spectrometry characterized PAF as the major inflammatory component coeluting with iPC. Oxidatively fragmented phospholipids and iPC are markers of free radical generation in CCl4-intoxicated liver, but PAF generation by activated hepatic cells generated the inflammatory agent.
Asunto(s)
Tetracloruro de Carbono/metabolismo , Diterpenos , Mediadores de Inflamación/metabolismo , Hígado/metabolismo , Fosfatidilcolinas/metabolismo , Factor de Activación Plaquetaria/metabolismo , Glicoproteínas de Membrana Plaquetaria/metabolismo , Receptores de Superficie Celular , Receptores Acoplados a Proteínas G , Animales , Tetracloruro de Carbono/toxicidad , Línea Celular , Cromatografía Líquida de Alta Presión , Colorantes Fluorescentes/metabolismo , Depuradores de Radicales Libres/farmacología , Radicales Libres/metabolismo , Fura-2/análogos & derivados , Fura-2/metabolismo , Ginkgólidos , Humanos , Inflamación/metabolismo , Lactonas/farmacología , Lipoproteínas LDL/metabolismo , Oxidación-Reducción , Fosfatidilcolinas/química , Fosfolipasas A/farmacología , Fosfolipasas A1 , Factor de Activación Plaquetaria/química , Ratas , Proteínas Recombinantes/metabolismoRESUMEN
Synthetic high affinity peroxisome proliferator-activated receptor (PPAR) agonists are known, but biologic ligands are of low affinity. Oxidized low density lipoprotein (oxLDL) is inflammatory and signals through PPARs. We showed, by phospholipase A(1) digestion, that PPARgamma agonists in oxLDL arise from the small pool of alkyl phosphatidylcholines in LDL. We identified an abundant oxidatively fragmented alkyl phospholipid in oxLDL, hexadecyl azelaoyl phosphatidylcholine (azPC), as a high affinity ligand and agonist for PPARgamma. [(3)H]azPC bound recombinant PPARgamma with an affinity (K(d)((app)) approximately 40 nm) that was equivalent to rosiglitazone (BRL49653), and competition with rosiglitazone showed that binding occurred in the ligand-binding pocket. azPC induced PPRE reporter gene expression, as did rosiglitazone, with a half-maximal effect at 100 nm. Overexpression of PPARalpha or PPARgamma revealed that azPC was a specific PPARgamma agonist. The scavenger receptor CD36 is encoded by a PPRE-responsive gene, and azPC enhanced expression of CD36 in primary human monocytes. We found that anti-CD36 inhibited azPC uptake, and it inhibited PPRE reporter induction. Results with a small molecule phospholipid flippase mimetic suggest azPC acts intracellularly and that cellular azPC accumulation was efficient. Thus, certain alkyl phospholipid oxidation products in oxLDL are specific, high affinity extracellular ligands and agonists for PPARgamma that induce PPAR-responsive genes.