RESUMEN
Mutations in the gene that encodes the principal l-carnitine transporter, OCTN2, can lead to a reduced intracellular l-carnitine pool and the disease Primary Carnitine Deficiency. l-Carnitine supplementation is used therapeutically to increase intracellular l-carnitine. As AMPK and insulin regulate fat metabolism and substrate uptake, we hypothesized that AMPK-activating compounds and insulin would increase l-carnitine uptake in C2C12 myotubes. The cells express all three OCTN transporters at the mRNA level, and immunohistochemistry confirmed expression at the protein level. Contrary to our hypothesis, despite significant activation of PKB and 2DG uptake, insulin did not increase l-carnitine uptake at 100 nM. However, l-carnitine uptake was modestly increased at a dose of 150 nM insulin. A range of AMPK activators that increase intracellular calcium content [caffeine (10 mM, 5 mM, 1 mM, 0.5 mM), A23187 (10 µM)], inhibit mitochondrial function [sodium azide (75 µM), rotenone (1 µM), berberine (100 µM), DNP (500 µM)], or directly activate AMPK [AICAR (250 µM)] were assessed for their ability to regulate l-carnitine uptake. All compounds tested significantly inhibited l-carnitine uptake. Inhibition by caffeine was not dantrolene (10 µM) sensitive despite dantrolene inhibiting caffeine-mediated calcium release. Saturation curve analysis suggested that caffeine did not competitively inhibit l-carnitine transport. To assess the potential role of AMPK in this process, we assessed the ability of the AMPK inhibitor Compound C (10 µM) to rescue the effect of caffeine. Compound C offered a partial rescue of l-carnitine uptake with 0.5 mM caffeine, suggesting that AMPK may play a role in the inhibitory effects of caffeine. However, caffeine likely inhibits l-carnitine uptake by alternative mechanisms independently of calcium release. PKA activation or direct interference with transporter function may play a role.
Asunto(s)
Carnitina/antagonistas & inhibidores , Activadores de Enzimas/farmacología , Mioblastos/efectos de los fármacos , Proteínas de Transporte de Catión Orgánico/metabolismo , Proteínas Quinasas Activadas por AMP/genética , Proteínas Quinasas Activadas por AMP/metabolismo , Aminoimidazol Carboxamida/análogos & derivados , Aminoimidazol Carboxamida/farmacología , Animales , Berberina/farmacología , Transporte Biológico/efectos de los fármacos , Cafeína/farmacología , Calcimicina/farmacología , Calcio/metabolismo , Carnitina/metabolismo , Línea Celular , Dantroleno/farmacología , Activación Enzimática/efectos de los fármacos , Expresión Génica , Insulina/farmacología , Ratones , Mioblastos/citología , Mioblastos/enzimología , Proteínas de Transporte de Catión Orgánico/genética , Isoformas de Proteínas/agonistas , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Ribonucleótidos/farmacología , Rotenona/farmacología , Azida Sódica/farmacología , Miembro 5 de la Familia 22 de Transportadores de SolutosRESUMEN
High-affinity and selective antagonists that are able to block the actions of both endogenous and synthetic agonists of G protein-coupled receptors are integral to analysis of receptor function and to support suggestions of therapeutic potential. Although there is great interest in the potential of free fatty acid receptor 4 (FFA4) as a novel therapeutic target for the treatment of type II diabetes, the broad distribution pattern of this receptor suggests it may play a range of roles beyond glucose homeostasis in different cells and tissues. To date, a single molecule, 4-methyl-N-9H-xanthen-9-yl-benzenesulfonamide (AH-7614), has been described as an FFA4 antagonist; however, its mechanism of antagonism remains unknown. We synthesized AH-7614 and a chemical derivative and demonstrated these to be negative allosteric modulators (NAMs) of FFA4. Although these NAMs did inhibit FFA4 signaling induced by a range of endogenous and synthetic agonists, clear agonist probe dependence in the nature of allosteric modulation was apparent. Although AH-7614 did not antagonize the second long-chain free fatty acid receptor, free fatty acid receptor 1, the simple chemical structure of AH-7614 containing features found in many anticancer drugs suggests that a novel close chemical analog of AH-7614 devoid of FFA4 activity, 4-methyl-N-(9H-xanthen-9-yl)benzamide (TUG-1387), will also provide a useful control compound for future studies assessing FFA4 function. Using TUG-1387 alongside AH-7614, we show that endogenous activation of FFA4 expressed by murine C3H10T1/2 mesenchymal stem cells is required for induced differentiation of these cells toward a more mature, adipocyte-like phenotype.
Asunto(s)
Compuestos de Bifenilo/farmacología , Fenilpropionatos/farmacología , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/fisiología , Regulación Alostérica/efectos de los fármacos , Regulación Alostérica/fisiología , Animales , Línea Celular Tumoral , Relación Dosis-Respuesta a Droga , Células HEK293 , Humanos , Ratones Endogámicos C3H , Receptores Acoplados a Proteínas G/antagonistas & inhibidoresRESUMEN
Various foods are associated with effects against metabolic diseases such as insulin resistance and type 2 diabetes; however, their mechanisms of action are mostly unclear. Fatty acids may contribute by acting as precursors of signalling molecules or by direct activity on receptors. The medium- and long-chain NEFA receptor FFA1 (free fatty acid receptor 1, previously known as GPR40) has been linked to enhancement of glucose-stimulated insulin secretion, whereas FFA4 (free fatty acid receptor 4, previously known as GPR120) has been associated with insulin-sensitising and anti-inflammatory effects, and both receptors are reported to protect pancreatic islets and promote secretion of appetite and glucose-regulating hormones. Hypothesising that FFA1 and FFA4 mediate therapeutic effects of dietary components, we screened a broad selection of NEFA on FFA1 and FFA4 and characterised active compounds in concentration-response curves. Of the screened compounds, pinolenic acid, a constituent of pine nut oil, was identified as a relatively potent and efficacious dual FFA1/FFA4 agonist, and its suitability for further studies was confirmed by additional in vitro characterisation. Pine nut oil and free and esterified pure pinolenic acid were tested in an acute glucose tolerance test in mice. Pine nut oil showed a moderately but significantly improved glucose tolerance compared with maize oil. Pure pinolenic acid or ethyl ester gave robust and highly significant improvements of glucose tolerance. In conclusion, the present results indicate that pinolenic acid is a comparatively potent and efficacious dual FFA1/FFA4 agonist that exerts antidiabetic effects in an acute mouse model. The compound thus deserves attention as a potential active dietary ingredient to prevent or counteract metabolic diseases.
Asunto(s)
Grasas de la Dieta/farmacología , Ácidos Linolénicos/farmacología , Síndrome Metabólico/prevención & control , Receptores Acoplados a Proteínas G/genética , Animales , Diabetes Mellitus Tipo 2/prevención & control , Modelos Animales de Enfermedad , Prueba de Tolerancia a la Glucosa , Células HEK293 , Humanos , Insulina/sangre , Insulina/metabolismo , Resistencia a la Insulina , Secreción de Insulina , Islotes Pancreáticos/efectos de los fármacos , Islotes Pancreáticos/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Nueces/química , Pinus , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/metabolismoRESUMEN
Glucose-sensing (GS) behaviour in pancreatic ß-cells is dependent on ATP-sensitive K(+) channel (KATP) activity, which is controlled by the relative levels of the KATP ligands ATP and ADP, responsible for closing and opening KATP, respectively. However, the mechanism by which ß-cells transfer energy status from mitochondria to KATP, and hence to altered electrical excitability and insulin secretion, is presently unclear. Recent work has demonstrated a critical role for AMP-activated protein kinase (AMPK) in GS behaviour of cells. Electrophysiological recordings, coupled with measurements of gene and protein expression were made from rat insulinoma cells to investigate whether AMPK activity regulates this energy transfer process. Using the whole-cell recording configuration with sufficient intracellular ATP to keep KATP closed, raised AMPK activity induced GS electrical behaviour. This effect was prevented by the AMPK inhibitor, compound C and required a phosphotransfer process. Indeed, high levels of intracellular phosphocreatine or the presence of the adenylate kinase (AK) inhibitor AP5A blocked this action of AMPK. Using conditions that maximised AMPK-induced KATP opening, there was a significant increase in AK1, AK2 and UCP2 mRNA expression. Thus we propose that KATP opening in response to lowered glucose concentration requires AMPK activity, perhaps in concert with increased AK and UCP2 to enable mitochondrial-derived ADP signals to be transferred to plasma membrane KATP by phosphotransfer cascades.
Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Células Secretoras de Insulina/metabolismo , Canales de Potasio/metabolismo , Proteínas Quinasas Activadas por AMP/antagonistas & inhibidores , Proteínas Quinasas Activadas por AMP/genética , Adenilato Quinasa/genética , Adenilato Quinasa/metabolismo , Animales , Línea Celular Tumoral , Fosfatos de Dinucleósidos/farmacología , Canales Iónicos/genética , Canales Iónicos/metabolismo , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Fosforilación/fisiología , Canales de Potasio/genética , Pirazoles/farmacología , Pirimidinas/farmacología , Ratas , Proteína Desacopladora 2 , Vasoconstrictores/farmacologíaRESUMEN
Activation of mammalian target of rapamycin 1 (mTORC1) by nutrients, insulin and leptin leads to appetite suppression (anorexia). Contrastingly, increased AMP-activated protein kinase (AMPK) activity by ghrelin promotes appetite (orexia). However, the interplay between these mechanisms remains poorly defined. The relationship between the anorexigenic hormones, insulin and leptin, and the orexigenic hormone, ghrelin, on mTORC1 signalling was examined using S6 kinase phosphorylation as a marker for changes in mTORC1 activity in mouse hypothalamic GT1-7 cells. Additionally, the contribution of AMPK and mTORC1 signalling in relation to insulin-, leptin- and ghrelin-driven alterations to mouse hypothalamic agouti-related protein (AgRP) mRNA levels was examined. Insulin and leptin increase mTORC1 activity in a phosphoinositide-3-kinase (PI3K)- and protein kinase B (PKB)-dependent manner, compared to vehicle controls, whereas increasing AMPK activity inhibits mTORC1 activity and blocks the actions of the anorexigenic hormones. Ghrelin mediates an AMPK-dependent decrease in mTORC1 activity and increases hypothalamic AgRP mRNA levels, the latter effect being prevented by insulin in an mTORC1-dependent manner. In conclusion, mTORC1 acts as an integration node in hypothalamic neurons for hormone-derived PI3K and AMPK signalling and mediates at least part of the assimilated output of anorexigenic and orexigenic hormone actions in the hypothalamus.
Asunto(s)
Proteína Relacionada con Agouti/biosíntesis , Apetito/fisiología , Hipotálamo/metabolismo , Complejos Multiproteicos/metabolismo , ARN Mensajero/biosíntesis , Serina-Treonina Quinasas TOR/metabolismo , Proteínas Quinasas Activadas por AMP/metabolismo , Animales , Anorexia/metabolismo , Femenino , Masculino , Diana Mecanicista del Complejo 1 de la Rapamicina , Ratones , Ratones Endogámicos C57BLRESUMEN
The process of neurite extension after activation of the TrkA tyrosine kinase receptor by nerve growth factor (NGF) involves complex signaling pathways. Stimulation of sphingosine kinase 1 (SphK1), the enzyme that phosphorylates sphingosine to form sphingosine-1-phosphate (S1P), is part of the functional TrkA signaling repertoire. In this paper, we report that in PC12 cells and dorsal root ganglion neurons, NGF translocates SphK1 to the plasma membrane and differentially activates the S1P receptors S1P1 and S1P2 in a SphK1-dependent manner, as determined with specific inhibitors and small interfering RNA targeted to SphK1. NGF-induced neurite extension was suppressed by down-regulation of S1P1 expression with antisense RNA. Conversely, when overexpressed in PC12 cells, transactivation of S1P1 by NGF markedly enhanced neurite extension and stimulation of the small GTPase Rac, important for the cytoskeletal changes required for neurite extension. Concomitantly, differentiation down-regulated expression of S1P2 whose activation would stimulate Rho and inhibit neurite extension. Thus, differential transactivation of S1P receptors by NGF regulates antagonistic signaling pathways that modulate neurite extension.
Asunto(s)
Factor de Crecimiento Nervioso/metabolismo , Neuritas/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Activación Transcripcional , Animales , Ganglios Espinales/metabolismo , Células PC12 , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Ratas , Receptores Acoplados a Proteínas G/genética , Receptores Lisofosfolípidos , Proteínas de Unión al GTP rac/metabolismo , Proteínas de Unión al GTP rho/metabolismoRESUMEN
Overactive bladder syndrome (OBS) results from disturbances of bladder function. Bladder smooth muscle (detrusor) exhibits spontaneous rhythmic activity (tone) independent of neurogenic control, which is enhanced in patients with OBS. We have now uncovered a prominent role for the bioactive sphingolipid metabolite, sphingosine-1-phosphate (S1P), in regulating rabbit detrusor smooth muscle tone and contraction. S1P-induced contraction of detrusor muscle was dependent on stretch and intracellular calcium. Although detrusor expresses the S1P receptors S1P1 and S1P2, only S1P2 appeared to be involved in S1P-induced contraction, since SEW2871 (S1P1 agonist) and dihydro-S1P (potent agonist for all S1P receptors except S1P2) were poor contractile agents. In agreement, the S1P2 antagonist JTE013 inhibited S1P-induced contraction. The fast, transient muscle contraction (phasic) mediated by S1P was dependent on phospholipase C (PLC) whereas the slower, sustained contraction (tonic) was not. Surprisingly, the immunosuppressant FTY720-phosphate, an agonist for all S1P receptors except S1P2, had distinct contractile properties and also induced slow, sustained contraction. Thus, FTY720-phosphate and/or S1P may regulate calcium channels in an S1P receptor-independent manner. Collectively, our results demonstrate that S1P may regulate detrusor smooth muscle tone and suggest that dysregulation of complex S1P signaling might contribute to OBS.
Asunto(s)
Inmunosupresores/farmacología , Lisofosfolípidos/farmacología , Tono Muscular/efectos de los fármacos , Músculo Liso/metabolismo , Glicoles de Propileno/farmacología , Esfingosina/análogos & derivados , Animales , Calcio/metabolismo , Señalización del Calcio , Células Cultivadas , Femenino , Clorhidrato de Fingolimod , Immunoblotting , Contracción Muscular/efectos de los fármacos , Músculo Liso/citología , Conejos , Receptores de Lisoesfingolípidos/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Esfingosina/farmacología , Fosfolipasas de Tipo C/farmacologíaRESUMEN
Sphingosine-1-phosphate (S1P), a bioactive sphingolipid metabolite, is the ligand for five specific G protein-coupled receptors, named S1P(1) to S1P(5). In this study, we found that cross-communication between platelet-derived growth factor receptor and S1P(2) serves as a negative damper of PDGF functions. Deletion of the S1P(2) receptor dramatically increased migration of mouse embryonic fibroblasts toward S1P, serum, and PDGF but not fibronectin. This enhanced migration was dependent on expression of S1P(1) and sphingosine kinase 1 (SphK1), the enzyme that produces S1P, as revealed by downregulation of their expression with antisense RNA and small interfering RNA, respectively. Although S1P(2) deletion had no significant effect on tyrosine phosphorylation of the PDGF receptors or activation of extracellular signal-regulated kinase 1/2 or Akt induced by PDGF, it reduced sustained PDGF-dependent p38 phosphorylation and markedly enhanced Rac activation. Surprisingly, S1P(2)-null cells not only exhibited enhanced proliferation but also markedly increased SphK1 expression and activity. Conversely, reintroduction of S1P(2) reduced DNA synthesis and expression of SphK1. Thus, S1P(2) serves as a negative regulator of PDGF-induced migration and proliferation as well as SphK1 expression. Our results suggest that a complex interplay between PDGFR and S1P receptors determines their functions.
Asunto(s)
Movimiento Celular/efectos de los fármacos , Factor de Crecimiento Derivado de Plaquetas/farmacología , Receptores de Lisoesfingolípidos/metabolismo , Animales , Estructuras de la Membrana Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Quimiotaxis/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Femenino , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Fibroblastos/enzimología , Fibroblastos/metabolismo , Eliminación de Gen , Expresión Génica , Masculino , Ratones , Modelos Biológicos , Fosfotransferasas (Aceptor de Grupo Alcohol)/antagonistas & inhibidores , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Receptores de Lisoesfingolípidos/deficiencia , Receptores de Lisoesfingolípidos/genética , Proteínas de Unión al GTP rac/metabolismoRESUMEN
Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid metabolite that is known to mediate diverse cellular responses including cell growth, survival, and migration. Most of these effects have been attributed to its binding to a specific subfamily of G protein-coupled receptors (GPCR), namely S1P(1-5). Recent studies have suggested that S1P also plays a prominent role in the contraction of various types of smooth muscle. This review provides a brief overview of its role in this process and also highlights how S1P-dependent signaling serves as an important regulator of smooth muscle contraction.
Asunto(s)
Calcio/metabolismo , Lisofosfolípidos/fisiología , Músculo Liso/fisiología , Esfingosina/análogos & derivados , Esfingosina/fisiología , Animales , Proteínas Quinasas Dependientes de GMP Cíclico/fisiología , Humanos , Contracción Muscular/fisiología , Relajación Muscular/fisiología , Músculo Liso/metabolismo , Receptores de Lisoesfingolípidos/fisiologíaRESUMEN
The free fatty acid receptor 4 (FFA4 or GPR120) has appeared as an interesting potential target for the treatment of metabolic disorders. At present, most FFA4 ligands are carboxylic acids that are assumed to mimic the endogenous long-chain fatty acid agonists. Here, we report preliminary structure-activity relationship studies of a previously disclosed nonacidic sulfonamide FFA4 agonist. Mutagenesis studies indicate that the compounds are orthosteric agonists despite the absence of a carboxylate function. The preferred compounds showed full agonist activity on FFA4 and complete selectivity over FFA1, although a significant fraction of these noncarboxylic acids also showed partial antagonistic activity on FFA1. Studies in normal and diet-induced obese (DIO) mice with the preferred compound 34 showed improved glucose tolerance after oral dosing in an oral glucose tolerance test. Chronic dosing of 34 in DIO mice resulted in significantly increased insulin sensitivity and a moderate but significant reduction in bodyweight, effects that were also present in mice lacking FFA1 but absent in mice lacking FFA4.
Asunto(s)
Hipoglucemiantes/química , Hipoglucemiantes/farmacología , Receptores Acoplados a Proteínas G/agonistas , Sulfonamidas/química , Sulfonamidas/farmacología , Animales , Línea Celular , Prueba de Tolerancia a la Glucosa , Humanos , Resistencia a la Insulina , Ratones , Ratones Endogámicos C57BL , Ratones Obesos , Modelos Moleculares , Simulación del Acoplamiento Molecular , Receptores Acoplados a Proteínas G/metabolismo , Relación Estructura-ActividadRESUMEN
The bioactive sphingolipid sphingosine-1-phosphate (S1P) that is increased in airways of asthmatic subjects markedly induced contraction of human airway smooth muscle (HASM) cells embedded in collagen matrices in a Gi-independent manner. Dihydro-S1P, which binds to S1P receptors, also stimulated contractility. S1P induced formation of stress fibers, contraction of individual HASM cells, and stimulated myosin light chain phosphorylation, which was inhibited by the Rho-associated kinase inhibitor Y-27632. S1P-stimulated HASM cell contractility was independent of the ERK1/2 and PKC signaling pathways, important regulators of airway smooth muscle contraction. However, removal of extracellular calcium completely blocked S1P-mediated contraction and Y-27632 reduced it. S1P also induced calcium mobilization that was not desensitized by repeated additions. Pretreatment with thapsigargin to deplete InsP3-sensitive calcium stores partially blocked increases in [Ca2+]i induced by S1P, yet did not inhibit S1P-stimulated contraction. In sharp contrast, the L-type calcium channel blocker verapamil markedly decreased S1P-induced HASM cell contraction, supporting a role for calcium influx from extracellular sources. Collectively, our results suggest that S1P may regulate HASM contractility, important in the pathobiology of asthma.
Asunto(s)
Lisofosfolípidos , Contracción Muscular , Músculo Liso/fisiología , Fenómenos Fisiológicos Respiratorios , Esfingosina/análogos & derivados , Esfingosina/farmacología , Calcio/metabolismo , Células Cultivadas , Colágeno , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/fisiología , Humanos , Péptidos y Proteínas de Señalización Intracelular , Transporte Iónico , Proteínas Quinasas Activadas por Mitógenos/fisiología , Músculo Liso/efectos de los fármacos , Músculo Liso/metabolismo , Proteína Quinasa C/fisiología , Proteínas Serina-Treonina Quinasas/fisiología , Transducción de Señal , Tráquea/citología , Proteínas de Unión al GTP rho/fisiología , Quinasas Asociadas a rhoRESUMEN
Dietary free fatty acids (FFAs), such as ω-3 fatty acids, regulate metabolic and anti-inflammatory processes, with many of these effects attributed to FFAs interacting with a family of G protein-coupled receptors. Selective synthetic ligands for free fatty acid receptors (FFA1-4) have consequently been developed as potential treatments for type 2 diabetes (T2D). In particular, clinical studies show that Fasiglifam, an agonist of the long-chain FFA receptor, FFA1, improved glycemic control and reduced HbA1c levels in T2D patients, with a reduced risk of hypoglycemia. However, this ligand was removed from clinical trials due to potential liver toxicity and determining if this is a target or a ligand-specific feature is now of major importance. Pre-clinical studies also show that FFA4 agonism increases insulin sensitivity, induces weight loss, and reduces inflammation and the metabolic and anti-inflammatory effects of short chain fatty acids (SCFAs) are linked with FFA2 and FFA3 activation. In this review, we therefore show that FFA receptor agonism is a potential clinical target for T2D treatment and discuss ongoing drug development programs within industry and academia aimed at improving the safety and effectiveness of these potential treatments.
RESUMEN
Leptin activates multiple signaling pathways in cells, including the phosphatidylinositol 3-kinase pathway, indicating a degree of cross-talk with insulin signaling. The exact mechanisms by which leptin alters this signaling pathway and how it relates to functional outputs are unclear at present. A previous study has established that leptin inhibits the activity of the phosphatase PTEN (phosphatase and tensin homolog deleted on chromosome 10), an important tumor suppressor and modifier of phosphoinositide signaling. In this study we demonstrate that leptin phosphorylates multiple sites on the C-terminal tail of PTEN in hypothalamic and pancreatic beta-cells, an action not replicated by insulin. Inhibitors of the protein kinases CK2 and glycogen synthase kinase 3 (GSK3) block leptin-mediated PTEN phosphorylation. PTEN phosphorylation mutants reveal the critical role these sites play in transmission of the leptin signal to F-actin depolymerization. CK2 and GSK3 inhibitors also prevent leptin-mediated F-actin depolymerization and consequent ATP-sensitive K(+) channel opening. GSK3 kinase activity is inhibited by insulin but not leptin in hypothalamic cells. Both hormones increase N-terminal GSK3 serine phosphorylation, but in hypothalamic cells this action of leptin is transient. Leptin, not insulin, increases GSK3 tyrosine phosphorylation in both cell types. These results demonstrate a significant role for PTEN in leptin signal transmission and identify GSK3 as a potential important signaling node contributing to divergent outputs for these hormones.
Asunto(s)
Actinas/metabolismo , Adenosina Trifosfato/metabolismo , Leptina/farmacología , Fosfohidrolasa PTEN/metabolismo , Canales de Potasio/metabolismo , Secuencia de Aminoácidos , Animales , Quinasa de la Caseína II/metabolismo , Línea Celular , Activación Enzimática , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Mutación/genética , Fosfohidrolasa PTEN/química , Fosfohidrolasa PTEN/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación/efectos de los fármacosRESUMEN
The bioactive lysophospholipids, primarily lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P), are recent additions to the list of potent mediators of tissue repair and wound healing. In this review, we highlight the diverse actions of LPA and S1P on many types of cells involved in the wound healing process, with special emphasis on their regulation of fibroblasts. The effects of LPA and S1P are principally mediated via specific cell surface receptors. Important signaling pathways downstream of these receptors and the importance of TGFbeta and S1P cross-talk for wound healing are also discussed. Moreover, specific agonists and antagonists of the lysophospholipid receptors may be useful for the treatment of wounds and abnormal wound healing.
Asunto(s)
Fibroblastos/fisiología , Lisofosfolípidos/fisiología , Cicatrización de Heridas/fisiología , Animales , Movimiento Celular/fisiología , Humanos , Fosforilación , Receptor Cross-Talk/fisiología , Receptores de Lisoesfingolípidos/fisiología , Esfingosina/análogos & derivados , Esfingosina/fisiología , Factor de Crecimiento Transformador beta/fisiologíaRESUMEN
Mast cells play a central role in inflammatory and immediate-type allergic reactions by secreting a variety of biologically active substances, including sphingosine-1 phosphate (S1P). Sphingosine kinase 1 (SphK1) and formation of S1P, which leads to transactivation of S1P receptors and their downstream signaling pathways, regulates mast-cell functions initiated by cross-linking of the high-affinity immunoglobulin E (IgE) receptor FcepsilonRI. Surprisingly, overexpression of SphK1 in rat basophilic leukemia (RBL)-2H3 mast cells impaired degranulation as well as migration toward antigen. These effects were reversed by serum withdrawal, yet the increased formation and secretion of S1P were the same as in the presence of serum. Nonetheless, serum increased localization of SphK1 at the plasma membrane. This restricted formation of S1P induced internalization and desensitization of S1P receptors on the surface of mast cells as determined by confocal immunofluorescence microscopy, aberrant S1P receptor signaling, and lack of S1P receptor coupling to G proteins. Serum starvation, which significantly reduced membrane-associated SphK1 activity, restored S1P receptor functions. Our results have important implications for mast-cell migration and degranulation as well as for the biologic functions of the S1P receptors on cells that are circulating in the bloodstream.
Asunto(s)
Mastocitos/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/biosíntesis , Fosfotransferasas (Aceptor de Grupo Alcohol)/química , Receptores de Lisoesfingolípidos/metabolismo , Animales , Western Blotting , Calcio/metabolismo , Línea Celular Tumoral , Membrana Celular/metabolismo , Movimiento Celular , Quimiotaxis , Reactivos de Enlaces Cruzados/farmacología , Medio de Cultivo Libre de Suero/farmacología , Regulación hacia Abajo , Subunidad alfa de la Proteína de Unión al GTP Gi2 , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/metabolismo , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Inmunoglobulina E/química , Inflamación , Ligandos , Microscopía Confocal , Microscopía Fluorescente , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Fenotipo , Fosforilación , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Transporte de Proteínas , Proteínas Proto-Oncogénicas/metabolismo , Ratas , Receptores de IgE/química , Transducción de Señal , Factores de Tiempo , Transfección , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
In this study, we have characterized the differential effects on inhibitory adenosine receptor (AR) trafficking of disrupting predicted sites for palmitoylation and phosphorylation within each receptor's carboxyl terminus. While a Cys(302,305)Ala-mutated rat A(3)AR mutant internalizes significantly faster than the wild-type (WT) receptor in response to agonist exposure, analogous mutation of the human A(1)AR (Cys(309)Ala) had no effect on receptor internalization. Moreover, unlike the WT A(3)AR, the entire pool of internalized mutant A(3)AR is able to recycle back to the plasma membrane following agonist removal. These properties do not reflect utilization of an alternative trafficking pathway, as internalized WT and mutant A(3)ARs both accumulate into transferrin receptor-positive endosomal compartments. However, receptor accumulation into endosomes is dependent upon prior G-protein-coupled receptor kinase (GRK)-mediated phosphorylation of the receptor's carboxyl terminus, as replacement of the carboxyl-terminal domain of the human A(1)AR with the 14 GRK-phosphorylated amino acids of the rat A(3)AR confers rapid agonist-mediated endosomal accumulation of the resulting chimeric A(1)CT3AR. Sensitivity to GRK-mediated phosphorylation also dictates the distinct redistribution of arrestin3 observed upon agonist exposure. Thus, while the nonphosphorylated A(1)AR redistributes arrestin3 from the cytoplasm to punctate clusters at the plasma membrane, GRK-phosphorylated WT and Cys(302,305)Ala-mutated A(3)ARs, as well as the A(1)CT3AR chimera, each induce the redistribution of arrestin3 into punctate accumulations both at the plasma membrane and within the cytoplasm. Neither the human A(1)AR nor the rat A(3)AR colocalized with arrestin3 under basal or agonist-stimulated conditions. Together, these results demonstrate that inhibitory AR-mediated changes in arrestin3 distribution are subtype-specific, with specificity correlating with the sensitivity of the receptor's carboxyl-terminal domain to GRK phosphorylation. In the case of the rat A(3)AR, sensitivity to GRK-mediated internalization appears to be regulated in part by the integrity of putative palmitate attachment sites upstream of its GRK phosphoacceptor sites.
Asunto(s)
Arrestinas/metabolismo , Agonistas del Receptor Purinérgico P1 , Receptores Purinérgicos P1/metabolismo , Alanina/genética , Animales , Células CHO , Línea Celular , Cricetinae , Proteínas Quinasas Dependientes de AMP Cíclico/fisiología , Cisteína/genética , Endosomas/genética , Endosomas/metabolismo , Proteínas Fluorescentes Verdes , Humanos , Cinética , Proteínas Luminiscentes/biosíntesis , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Mutagénesis Sitio-Dirigida , Ácido Palmítico/metabolismo , Fosforilación , Estructura Terciaria de Proteína/genética , Transporte de Proteínas/genética , Ratas , Receptor de Adenosina A3 , Receptores Purinérgicos P1/química , Receptores Purinérgicos P1/genética , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Transfección , Quinasas de Receptores Adrenérgicos betaRESUMEN
Here we demonstrate that phosphorylation of the sphingosine 1-phosphate (SSP) receptor "endothelial differentiation gene 1" (EDG1 or S1P(1)) receptor is increased in response to either SSP or phorbol 12-myristate 13-acetate (PMA) exposure but not lysophosphatidic acid. Phosphoamino acid analysis demonstrated that SSP stimulated the accumulation of phosphoserine and phosphothreonine but not phosphotyrosine. An inhibitor of PMA-stimulated EDG1 phosphorylation failed to block SSP-stimulated phosphorylation. Additionally, removal of 12 amino acids from the carboxyl terminus of EDG1 specifically reduced SSP- but not PMA-stimulated phosphorylation, suggesting that SSP and PMA increase EDG1 phosphorylation via distinct mechanisms. In vitro assays revealed that G-protein-coupled receptor kinase 2 may be at least partially responsible for SSP-stimulated EDG1 phosphorylation observed in intact cells. In addition, phosphorylation by PMA and SSP were associated with a loss of EDG1 from the cell surface by distinct mechanisms. Removal of 12 residues from the carboxyl terminus of EDG1 completely inhibited SSP-mediated internalization, suggesting that this domain dictates susceptibility to receptor internalization while retaining sensitivity to SSP-stimulated phosphorylation. Thus, we conclude that (a) EDG1 phosphorylation and internalization are controlled via independent mechanisms by agonist occupation of the receptor and protein kinase C activation, and (b) although determinants within the receptor's carboxyl-terminal tail conferring EDG1 sensitivity to agonist-mediated internalization and G-protein-coupled receptor kinase phosphorylation exhibit a degree of overlap, the two phenomena are separable.