RESUMEN
OBJECTIVES: Drug overdose deaths in Connecticut increasingly involve a growing number of fentanyl analogs and other novel nonfentanyl synthetic opioids (ie, novel synthetics). Current postmortem toxicology testing methods often lack the sophistication needed to detect these compounds. We examined how improved toxicology testing of fatal drug overdoses can determine the prevalence and rapidly evolving trends of novel synthetics. METHODS: From 2016 to June 2019, the Connecticut Office of the Chief Medical Examiner increased its scope of toxicology testing of suspected drug overdose deaths in Connecticut from basic to enhanced toxicology testing to detect novel synthetics. The toxicology laboratory also expanded its testing panels during this time. We analyzed toxicology results to identify and quantify the involvement of novel synthetics over time. RESULTS: From 2016 to June 2019, 3204 drug overdose deaths received enhanced toxicology testing; novel synthetics were detected in 174 (5.4%) instances. Ten different novel synthetics were detected with 205 total occurrences. Of 174 overdose deaths with a novel synthetic detected, most had 1 (n = 146, 83.9%) or 2 (n = 26, 14.9%) novel synthetics detected, with a maximum of 4 novel synthetics detected. Para-fluorobutyrylfentanyl/FIBF, furanylfentanyl, and U-47700 were most identified overall, but specific novel synthetics came in and out of prominence during the study period, and the variety of novel synthetics detected changed from year to year. CONCLUSIONS: Enhanced toxicology testing for drug overdose deaths is effective in detecting novel synthetics that are not identified through basic toxicology testing. Identifying emerging novel synthetics allows for a timely and focused response to potential drug outbreaks and illustrates the changing drug market.
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Fentanilo/análisis , Sobredosis de Opiáceos/sangre , Toxicología/normas , Connecticut/epidemiología , Fentanilo/sangre , Humanos , Sobredosis de Opiáceos/diagnóstico , Sobredosis de Opiáceos/epidemiología , Trastornos Relacionados con Sustancias/sangre , Trastornos Relacionados con Sustancias/diagnóstico , Trastornos Relacionados con Sustancias/epidemiología , Toxicología/métodos , Toxicología/estadística & datos numéricosRESUMEN
Sphingosine Kinase-2 (Sphk2) is responsible for the production of the bioactive lipid Sphingosine-1 Phosphate, a key regulator of tissue repair. Here we address the in vivo significance of Sphingosine Kinase -2 in renal inflammation/fibrosis in response to unilateral ureteral obstruction using both genetic and pharmacological strategies. Obstructed kidneys of Sphk2-/- mice showed reduced renal damage and diminished levels of the renal injury markers TGFß1 and αSMA when compared to wild type controls. We found a consistently significant increase in anti-inflammatory (M2) macrophages in obstructed Sphk2-/- kidneys by flow cytometry and a decrease in mRNA levels of the inflammatory cytokines, MCP1, TNFα, CXCL1 and ILß1, suggesting an anti-inflammatory bias in the absence of Sphk2. Indeed, metabolic profiling showed that the pro-inflammatory glycolytic pathway is largely inactive in Sphk2-/- bone marrow-derived macrophages. Furthermore, treatment with the M2-promoting cytokines IL-4 or IL-13 demonstrated that macrophages lacking Sphk2 polarized more efficiently to the M2 phenotype than wild type cells. Bone marrow transplant studies indicated that expression of Sphk2-/- on either the hematopoietic or parenchymal cells did not fully rescue the pro-healing phenotype, confirming that both infiltrating M2-macrophages and the kidney microenvironment contribute to the damaging Sphk2 effects. Importantly, obstructed kidneys from mice treated with an Sphk2 inhibitor recapitulated findings in the genetic model. These results demonstrate that reducing Sphk2 activity by genetic or pharmacological manipulation markedly decreases inflammatory and fibrotic responses to obstruction, resulting in diminished renal injury and supporting Sphk2 as a novel driver of the pro-inflammatory macrophage phenotype.
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Macrófagos/fisiología , Nefritis Intersticial/enzimología , Fosfotransferasas (Aceptor de Grupo Alcohol)/fisiología , Actinas/biosíntesis , Actinas/genética , Animales , Microambiente Celular , Citocinas/biosíntesis , Citocinas/genética , Fibrosis , Regulación de la Expresión Génica/inmunología , Glucólisis , Riñón/enzimología , Riñón/patología , Lisofosfolípidos/sangre , Lisofosfolípidos/fisiología , Activación de Macrófagos , Masculino , Ratones , Ratones Endogámicos C57BL , Nefritis Intersticial/etiología , Nefritis Intersticial/inmunología , Nefritis Intersticial/patología , Fosfotransferasas (Aceptor de Grupo Alcohol)/antagonistas & inhibidores , Fosfotransferasas (Aceptor de Grupo Alcohol)/deficiencia , Isoformas de Proteínas/fisiología , Inhibidores de Proteínas Quinasas/farmacología , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Esfingosina/análogos & derivados , Esfingosina/sangre , Esfingosina/fisiología , Factor de Crecimiento Transformador beta1/biosíntesis , Factor de Crecimiento Transformador beta1/genética , Obstrucción Ureteral/complicacionesRESUMEN
PURPOSE: The S1P signaling pathway represents an important potential target for the modulation of tissue inflammation/injury. The immunomodulator FTY720, also known as fingolimod, is a potent agonist for multiple S1P receptors that was approved by the Food and Drug Administration to treat multiple sclerosis. We examined the therapeutic role of FTY720 for renal injury secondary to unilateral ureteral obstruction. MATERIALS AND METHODS: CB57BL/6 mice underwent a sham procedure or unilateral ureteral obstruction and were treated with FTY720 by gavage for 1, 3 and 5 days. Control groups received vehicle. Ligated and unligated renal tissue was examined for histopathological changes, inflammatory and fibrotic markers, TGF-ß1, α-SMA, and macrophage infiltration by Western blot and immunohistochemistry. Proinflammatory and profibrotic cytokines were profiled by quantitative reverse transcriptase-polymerase chain reaction. RESULTS: Pathological evaluation revealed that FTY720 treatment resulted in a significant reduction in inflammatory infiltration in obstructed kidneys compared to controls. Immunohistochemical and Western blot showed that TGF-ß1 and α-SMA protein levels were similarly decreased, as was macrophage infiltration into the renal interstitial space, compared to untreated mice. In agreement with these observations quantitative reverse transcriptase-polymerase chain reaction revealed that inflammatory and fibrotic cytokines (MCP-1, IL-1ß, CXCL1, TNF-α and TGF-ß1) were also significantly decreased in the FTY720 group. CONCLUSIONS: This study suggests that in a murine ureteral obstruction model FTY720 significantly inhibited the production of inflammatory cytokines and factors regulating interstitial fibrosis and extracellular matrix accumulation. These findings were associated with decreased evidence of renal injury on pathological examination, suggesting that FTY720 or related compounds may be valuable modulators of obstruction induced renal injury.
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Inmunosupresores/uso terapéutico , Inflamación/prevención & control , Riñón/patología , Glicoles de Propileno/uso terapéutico , Esfingosina/análogos & derivados , Obstrucción Ureteral/tratamiento farmacológico , Obstrucción Ureteral/patología , Animales , Western Blotting , Modelos Animales de Enfermedad , Femenino , Fibrosis , Clorhidrato de Fingolimod , Inmunohistoquímica , Masculino , Ratones , Ratones Endogámicos C57BL , Esfingosina/uso terapéutico , Factor de Crecimiento Transformador beta1/metabolismoRESUMEN
GPCR inhibitors are highly prevalent in modern therapeutics. However, interference with complex GPCR regulatory mechanisms leads to both therapeutic efficacy and adverse effects. Recently, the sphingosine-1-phosphate (S1P) receptor inhibitor FTY720 (also known as Fingolimod), which induces lymphopenia and prevents neuroinflammation, was adopted as a disease-modifying therapeutic in multiple sclerosis. Although highly efficacious, dose-dependent increases in adverse events have tempered its utility. We show here that FTY720P induces phosphorylation of the C-terminal domain of S1P receptor 1 (S1P1) at multiple sites, resulting in GPCR internalization, polyubiquitinylation, and degradation. We also identified the ubiquitin E3 ligase WWP2 in the GPCR complex and demonstrated its requirement in FTY720-induced receptor degradation. GPCR degradation was not essential for the induction of lymphopenia, but was critical for pulmonary vascular leak in vivo. Prevention of receptor phosphorylation, internalization, and degradation inhibited vascular leak, which suggests that discrete mechanisms of S1P receptor regulation are responsible for the efficacy and adverse events associated with this class of therapeutics.
Asunto(s)
Síndrome de Fuga Capilar/fisiopatología , Glicoles de Propileno/toxicidad , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Lisoesfingolípidos/metabolismo , Esfingosina/análogos & derivados , Animales , Relación Dosis-Respuesta a Droga , Endocitosis , Clorhidrato de Fingolimod , Técnicas de Sustitución del Gen , Linfopenia/inducido químicamente , Lisofosfolípidos/fisiología , Ratones , Organofosfatos/farmacología , Péptido Hidrolasas/metabolismo , Fosforilación/efectos de los fármacos , Glicoles de Propileno/farmacología , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Estructura Terciaria de Proteína , Edema Pulmonar/inducido químicamente , Edema Pulmonar/fisiopatología , Receptores de Lisoesfingolípidos/genética , Proteínas Recombinantes de Fusión/fisiología , Esfingosina/farmacología , Esfingosina/fisiología , Esfingosina/toxicidad , Receptores de Esfingosina-1-Fosfato , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/fisiología , Ubiquitinación/efectos de los fármacosRESUMEN
OBJECTIVE: Sphingomyelin deposition and metabolism occurs in the atherosclerotic plaque, leading to the formation of sphingosine-1-phosphate (S1P), which activates G protein-coupled receptors to regulate vascular and immune cells. The role of S1P receptors in atherosclerosis has not been examined. METHODS AND RESULTS: We tested the hypothesis that S1P receptor-2 (S1PR2) regulates atherosclerosis. Apoe(-/-) S1pr2(-/-) mice showed greatly attenuated atherosclerosis compared with the Apoe(-/-) mice. Bone marrow transplant experiments indicate that S1PR2 function in the hematopoietic compartment is critical. S1PR2 is expressed in bone marrow-derived macrophages and in macrophage-like foam cells in atherosclerotic plaques. Reduced macrophage-like foam cells were found in the atherosclerotic plaques of Apoe(-/-)S1pr2(-/-) mice, suggesting that S1PR2 retains macrophages in atherosclerotic plaques. Lipoprotein profiles, plasma lipids, and oxidized low-density lipoprotein uptake by bone marrow-derived macrophages were not altered by the S1pr2 genotype. In contrast, endotoxin-induced inflammatory cytokine (interleukin [IL]-1ß, IL-18) levels in the serum of S1PR2 knockout mice were significantly reduced. Furthermore, treatment of wild-type mice with S1PR2 antagonist JTE-013 suppressed IL-1ß and IL-18 levels in plasma. CONCLUSIONS: These data suggest that S1PR2 signaling in the plaque macrophage regulates macrophage retention and inflammatory cytokine secretion, thereby promoting atherosclerosis.
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Enfermedades de la Aorta/metabolismo , Aterosclerosis/metabolismo , Inflamación/metabolismo , Macrófagos/metabolismo , Receptores de Lisoesfingolípidos/metabolismo , Animales , Enfermedades de la Aorta/etiología , Enfermedades de la Aorta/genética , Enfermedades de la Aorta/inmunología , Enfermedades de la Aorta/patología , Apolipoproteínas E/deficiencia , Apolipoproteínas E/genética , Aterosclerosis/etiología , Aterosclerosis/genética , Aterosclerosis/inmunología , Aterosclerosis/patología , Trasplante de Médula Ósea , Modelos Animales de Enfermedad , Endotoxinas , Inflamación/etiología , Inflamación/genética , Inflamación/inmunología , Inflamación/patología , Mediadores de Inflamación/sangre , Interleucina-18/sangre , Interleucina-1beta/sangre , Lípidos/sangre , Lipoproteínas/sangre , Lipoproteínas LDL/sangre , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Pirazoles/farmacología , Piridinas/farmacología , Receptores de Lisoesfingolípidos/antagonistas & inhibidores , Receptores de Lisoesfingolípidos/deficiencia , Receptores de Lisoesfingolípidos/genética , Receptores de Esfingosina-1-FosfatoRESUMEN
The sphingosine 1-phosphate receptor 1 (S1P(1)) promotes lymphocyte egress from lymphoid organs. Previous work showed that agonist-induced internalization of this G protein-coupled receptor correlates with inhibition of lymphocyte egress and results in lymphopenia. However, it is unclear if S1P(1) internalization is necessary for this effect. We characterize a knockin mouse (S1p1r(S5A/S5A)) in which the C-terminal serine-rich S1P(1) motif, which is important for S1P(1) internalization but dispensable for S1P(1) signaling, is mutated. T cells expressing the mutant S1P(1) showed delayed S1P(1) internalization and defective desensitization after agonist stimulation. Mutant mice exhibited significantly delayed lymphopenia after S1P(1) agonist administration or disruption of the vascular S1P gradient. Adoptive transfer experiments demonstrated that mutant S1P(1) expression in lymphocytes, rather than endothelial cells, facilitated this delay in lymphopenia. Thus, cell-surface residency of S1P(1) on T cells is a primary determinant of lymphocyte egress kinetics in vivo.
Asunto(s)
Membrana Celular/metabolismo , Quimiotaxis de Leucocito , Receptores de Lisoesfingolípidos/metabolismo , Linfocitos T/citología , Linfocitos T/metabolismo , Sustitución de Aminoácidos/genética , Animales , Secuencia de Bases , Membrana Celular/efectos de los fármacos , Quimiotaxis de Leucocito/efectos de los fármacos , Endocitosis/efectos de los fármacos , Clorhidrato de Fingolimod , Cinética , Linfopenia/genética , Linfopenia/patología , Lisofosfolípidos/agonistas , Lisofosfolípidos/metabolismo , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Proteínas Mutantes/metabolismo , Glicoles de Propileno/farmacología , Receptores de Lisoesfingolípidos/genética , Esfingosina/agonistas , Esfingosina/análogos & derivados , Esfingosina/metabolismo , Esfingosina/farmacología , Linfocitos T/efectos de los fármacosRESUMEN
Sphingosine 1-phosphate (S1P), an abundant lipid mediator in plasma, regulates vascular and immune cells by activating S1P receptors. In this report, we investigated the mechanisms by which high plasma S1P levels are maintained in mice. We found that plasma S1P turns over rapidly with a half-life of approximately 15 minutes, suggesting the existence of a high-capacity biosynthetic source(s). Transplantation of bone marrow from wild-type to Sphk1(-/-)Sphk2(+/-) mice restored plasma S1P levels, suggesting that hematopoietic cells are capable of secreting S1P into plasma. However, plasma S1P levels were not appreciably altered in mice that were thrombocytopenic, anemic, or leukopenic. Surprisingly, reconstitution of Sphk1(-/-)Sphk2(+/-) bone marrow cells into wild-type hosts failed to reduce plasma S1P, suggesting the existence of an additional, nonhematopoietic source for plasma S1P. Adenoviral expression of Sphk1 in the liver of Sphk1(-/-) mice restored plasma S1P levels. In vitro, vascular endothelial cells, but not hepatocytes, secreted S1P in a constitutive manner. Interestingly, laminar shear stress downregulated the expression of S1P lyase (Sgpl) and S1P phosphatase-1 (Sgpp1) while concomitantly stimulating S1P release from endothelial cells in vitro. Modulation of expression of endothelial S1P lyase with small interfering RNA and adenoviral expression altered S1P secretion, suggesting an important role played by this enzyme. These data suggest that the vascular endothelium, in addition to the hematopoietic system, is a major contributor of plasma S1P.
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Células de la Médula Ósea/metabolismo , Células Endoteliales/metabolismo , Endotelio Vascular/metabolismo , Lisofosfolípidos/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Esfingosina/análogos & derivados , Adenoviridae/genética , Aldehído-Liasas/genética , Aldehído-Liasas/metabolismo , Anemia/sangre , Anemia/inducido químicamente , Animales , Anticuerpos Monoclonales , Células de la Médula Ósea/enzimología , Células de la Médula Ósea/efectos de la radiación , Trasplante de Médula Ósea , Línea Celular , Línea Celular Tumoral , Células Cultivadas , Modelos Animales de Enfermedad , Células Endoteliales/enzimología , Endotelio Vascular/enzimología , Vectores Genéticos , Semivida , Humanos , Leucopenia/sangre , Hígado/enzimología , Hígado/metabolismo , Lisofosfolípidos/sangre , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fenilhidrazinas , Monoéster Fosfórico Hidrolasas/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/deficiencia , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Complejo GPIb-IX de Glicoproteína Plaquetaria/inmunología , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Esfingosina/sangre , Esfingosina/metabolismo , Estrés Mecánico , Trombocitopenia/sangre , Trombocitopenia/inmunología , Factores de Tiempo , Transducción Genética , Irradiación Corporal TotalRESUMEN
Sphingosine 1-phosphate (S1P), a multifunctional lipid mediator, regulates lymphocyte trafficking, vascular permeability, and angiogenesis by activation of the S1P1 receptor. This receptor is activated by FTY720-P, a phosphorylated derivative of the immunosuppressant and vasoactive compound FTY720. However, in contrast to the natural ligand S1P, FTY720-P appears to act as a functional antagonist, even though the mechanisms involved are poorly understood. In this study, we investigated the fate of endogenously expressed S1P1 receptor in agonist-activated human umbilical vein endothelial cells and human embryonic kidney 293 cells expressing green fluorescent protein-tagged S1P1. We show that FTY720-P is more potent than S1P at inducing receptor degradation. Pretreatment with an antagonist of S1P1, VPC 44116, prevented receptor internalization and degradation. FTY720-P did not induce degradation of internalization-deficient S1P1 receptor mutants. Further, small interfering RNA-mediated down-regulation of G protein-coupled receptor kinase-2 and beta-arrestins abolished FTY720-P-induced S1P1 receptor degradation. These data suggest that agonist-induced phosphorylation of S1P1 and subsequent endocytosis are required for FTY720-P-induced degradation of the receptor. S1P1 degradation is blocked by MG132, a proteasomal inhibitor. Indeed, FTY720-P strongly induced polyubiquitinylation of S1P1 receptor, whereas S1P at concentrations that induced complete internalization was not as efficient, suggesting that receptor internalization is required but not sufficient for ubiquitinylation and degradation. We propose that the ability of FTY720-P to target the S1P1 receptor to the ubiquitinylation and proteasomal degradation pathway may at least in part underlie its immunosuppressive and anti-angiogenic properties.
Asunto(s)
Inhibidores de la Angiogénesis/farmacología , Inmunosupresores/farmacología , Glicoles de Propileno/farmacología , Complejo de la Endopetidasa Proteasomal/metabolismo , Receptores de Lisoesfingolípidos/agonistas , Esfingosina/análogos & derivados , Ubiquitina/química , Línea Celular , Inhibidores Enzimáticos/farmacología , Clorhidrato de Fingolimod , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Leupeptinas/farmacología , Ligandos , Microscopía Confocal , Unión Proteica , Esfingosina/farmacologíaRESUMEN
Sphingosine 1-phosphate (S1P), produced by Sphks (sphingosine kinases), is a multifunctional lipid mediator that regulates immune cell trafficking and vascular development. Mammals maintain a large concentration gradient of S1P between vascular and extravascular compartments. Mechanisms by which S1P is released from cells and concentrated in the plasma are poorly understood. We recently demonstrated [Ancellin, Colmont, Su, Li, Mittereder, Chae, Stefansson, Liau and Hla (2002) J. Biol. Chem. 277, 6667-6675] that Sphk1 activity is constitutively secreted by vascular endothelial cells. In the present study, we show that among the five Sphk isoforms expressed in endothelial cells, the Sphk-1a isoform is selectively secreted in HEK-293 cells (human embryonic kidney cells) and human umbilical-vein endothelial cells. In sharp contrast, Sphk2 is not secreted. The exported Sphk-1a isoform is enzymatically active and produced sufficient S1P to induce S1P receptor internalization. Wild-type mouse plasma contains significant Sphk activity (179 pmol x min(-1) x g(-1)). In contrast, Sphk1-/- mouse plasma has undetectable Sphk activity and approx. 65% reduction in S1P levels. Moreover, human plasma contains enzymatically active Sphk1 (46 pmol x min(-1) x g(-1)). These results suggest that export of Sphk-1a occurs under physiological conditions and may contribute to the establishment of the vascular S1P gradient.
Asunto(s)
Lisofosfolípidos/biosíntesis , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Esfingosina/análogos & derivados , Animales , Células Cultivadas , Medios de Cultivo Condicionados , Endotelio Vascular/citología , Espacio Extracelular/metabolismo , Humanos , Espacio Intracelular/metabolismo , Isoenzimas/sangre , Isoenzimas/metabolismo , Lisofosfolípidos/sangre , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fosfotransferasas (Aceptor de Grupo Alcohol)/sangre , Transporte de Proteínas , Receptores de Lisoesfingolípidos/metabolismo , Esfingosina/biosíntesis , Esfingosina/sangreRESUMEN
The essential role of the sphingosine 1-phosphate (S1P) receptor S1P(1) in regulating lymphocyte trafficking was demonstrated with the S1P(1)-selective nanomolar agonist, SEW2871. Despite its lack of charged headgroup, the tetraaromatic compound SEW2871 binds and activates S1P(1) through a combination of hydrophobic and ion-dipole interactions. Both S1P and SEW2871 activated ERK, Akt, and Rac signaling pathways and induced S1P(1) internalization and recycling, unlike FTY720-phosphate, which induces receptor degradation. Agonism with receptor recycling is sufficient for alteration of lymphocyte trafficking by S1P and SEW2871. S1P(1) modeling and mutagenesis studies revealed that residues binding the S1P headgroup are required for kinase activation by both S1P and SEW2871. Therefore, SEW2871 recapitulates the action of S1P in all the signaling pathways examined and overlaps in interactions with key headgroup binding receptor residues, presumably replacing salt-bridge interactions with ion-dipole interactions.
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Evaluación Preclínica de Medicamentos , Sondas Moleculares/metabolismo , Oxadiazoles/farmacología , Receptores de Lisoesfingolípidos/agonistas , Receptores de Lisoesfingolípidos/metabolismo , Transducción de Señal/efectos de los fármacos , Tiofenos/farmacología , Animales , Sitios de Unión , Línea Celular , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Cricetinae , Activación Enzimática/efectos de los fármacos , Humanos , Ligandos , Lisofosfolípidos/química , Lisofosfolípidos/farmacología , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Modelos Moleculares , Sondas Moleculares/química , Sondas Moleculares/farmacología , Mutación/genética , Oxadiazoles/química , Fosforilación/efectos de los fármacos , Proteínas Serina-Treonina Quinasas/metabolismo , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-akt , Receptores de Lisoesfingolípidos/química , Receptores de Lisoesfingolípidos/genética , Esfingosina/análogos & derivados , Esfingosina/química , Esfingosina/farmacología , Tiofenos/química , Proteínas de Unión al GTP rac/metabolismoRESUMEN
PTEN, a tumor suppressor phosphatase, is important in the regulation of cell migration and invasion. Physiological regulation of PTEN (phosphatase and tensin homolog deleted on chromosome 10) by cell surface receptors has not been described. Here, we show that the bioactive lipid sphingosine 1-phosphate (S1P), which acts through the S1P2 receptor (S1P2R) G protein-coupled receptor (GPCR) to inhibit cell migration, utilizes PTEN as a signaling intermediate. S1P2R inhibition of cell migration is abrogated by dominant-negative PTEN expression. S1P was unable to efficiently inhibit the migration of Pten(DeltaloxP/DeltaloxP) mouse embryonic fibroblasts; however, the antimigratory effect was restored upon the expression of PTEN. S1P2R activation of Rho GTPase is not affected in Pten(DeltaloxP/DeltaloxP) cells, and dominant-negative Rho GTPase reversed S1P inhibition of cell migration in WT cells but not in Pten(DeltaloxP/DeltaloxP) cells, suggesting that PTEN acts downstream of the Rho GTPase. Ligand activation of the S1P2R receptor stimulated the coimmunoprecipitation of S1P2R and PTEN. Interestingly, S1P2R signaling increased PTEN phosphatase activity in membrane fractions. Furthermore, tyrosine phosphorylation of PTEN was stimulated by S1P2R signaling. These data suggest that the S1P2R receptor actively regulates the PTEN phosphatase by a Rho GTPase-dependent pathway to inhibit cell migration. GPCR regulation of PTEN maybe a general mechanism in signaling events of cell migration and invasion.
Asunto(s)
Monoéster Fosfórico Hidrolasas/metabolismo , Proteínas Tirosina Fosfatasas/metabolismo , Receptores de Lisoesfingolípidos/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Animales , Movimiento Celular/fisiología , Células Cultivadas , Humanos , Péptidos y Proteínas de Señalización Intracelular , Ratones , Fosfohidrolasa PTEN , Monoéster Fosfórico Hidrolasas/química , Fosforilación , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Tirosina Fosfatasas/química , Proteínas Tirosina Fosfatasas/deficiencia , Proteínas Tirosina Fosfatasas/genética , Receptores de Lisoesfingolípidos/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transducción de Señal , Transfección , Proteínas Supresoras de Tumor/química , Proteínas Supresoras de Tumor/deficiencia , Proteínas Supresoras de Tumor/genética , Tirosina/química , Proteínas de Unión al GTP rho/metabolismo , Quinasas Asociadas a rhoRESUMEN
OBJECTIVE: TWEAK, a member of the tumor necrosis factor superfamily, binds to the Fn14 receptor and stimulates angiogenesis in vivo. In this study, we investigated Fn14 gene expression in human endothelial cells (ECs) and examined the effect of TWEAK, added either alone or in combination with fibroblast growth factor-2 (FGF-2) or vascular endothelial growth factor-A (VEGF-A), on EC proliferation, migration, and survival in vitro. We also determined whether a soluble Fn14-Fc fusion protein could inhibit TWEAK biologic activity on ECs and investigated TWEAK signal transduction in ECs. METHODS AND RESULTS: We found that both FGF-2 and VEGF-A could induce Fn14 mRNA expression in ECs. TWEAK was a mitogen for ECs, and this proliferative activity could be inhibited by an Fn14-Fc decoy receptor. Furthermore, TWEAK treatment activated several intracellular signaling pathways in ECs and potentiated FGF-2--and VEGF-A--stimulated EC proliferation. TWEAK also had EC chemotactic activity, but it did not promote EC survival. CONCLUSIONS: These results indicate that TWEAK is an EC growth and migration factor but not a survival factor. TWEAK can also enhance both FGF-2 and VEGF-A mitogenic activity on ECs. Thus, TWEAK may act alone as well as in combination with FGF-2 or VEGF-A to regulate pathological angiogenesis.