RESUMEN
Phosphatidylinositol 3-phosphate (PI(3)P) is the predominant phosphoinositide species in early endosomes and autophagosomes, in which PI(3)P dictates traffic of these organelles. Phosphoinositide levels are tightly regulated by lipid-kinases and -phosphatases; however, a phosphatase that converts PI(3)P back to phosphatidylinositol in the endosomal and autophagosomal compartments is not fully understood. We investigated the subcellular distribution and functions of myotubularin-related protein-4 (MTMR4), which is distinct among other MTMRs in that it possesses a PI(3)P-binding FYVE domain, in lung alveolar epithelium-derived A549 cells. MTMR4 was localized mainly in late endosomes and autophagosomes. MTMR4 knockdown markedly suppressed the motility, fusion, and fission of PI(3)P-enriched structures, resulting in decreases in late endosomes, autophagosomes, and lysosomes, and enlargement of PI(3)P-enriched early and late endosomes. In amino acid- and serum-starved cells, MTMR4 knockdown decreased both autophagosomes and autolysosomes and markedly increased PI(3)P-containing autophagosomes and late endosomes, suggesting that the fusion with lysosomes of autophagosomes and late endosomes might be impaired. Notably, MTMR4 knockdown inhibited the nuclear translocation of starvation stress responsive transcription factor-EB (TFEB) with reduced expression of lysosome-related genes in starved cells. These findings indicate that MTMR4 is essential for the integrity of endocytic and autophagic pathways.
RESUMEN
We have recently demonstrated that the PI3K class II-α isoform (PI3K-C2α), which generates phosphatidylinositol 3-phosphate and phosphatidylinositol 3,4-bisphosphates, plays crucial roles in angiogenesis, by analyzing PI3K-C2α knock-out mice. The PI3K-C2α actions are mediated at least in part through its participation in the internalization of VEGF receptor-2 and sphingosine-1-phosphate receptor S1P1 and thereby their signaling on endosomes. TGFß, which is also an essential angiogenic factor, signals via the serine/threonine kinase receptor complex to induce phosphorylation of Smad2 and Smad3 (Smad2/3). SARA (Smad anchor for receptor activation) protein, which is localized in early endosomes through its FYVE domain, is required for Smad2/3 signaling. In the present study, we showed that PI3K-C2α knockdown nearly completely abolished TGFß1-induced phosphorylation and nuclear translocation of Smad2/3 in vascular endothelial cells (ECs). PI3K-C2α was necessary for TGFß-induced increase in phosphatidylinositol 3,4-bisphosphates in the plasma membrane and TGFß receptor internalization into the SARA-containing early endosomes, but not for phosphatidylinositol 3-phosphate enrichment or localization of SARA in the early endosomes. PI3K-C2α was also required for TGFß receptor-mediated formation of SARA-Smad2/3 complex. Inhibition of dynamin, which is required for the clathrin-dependent receptor endocytosis, suppressed both TGFß receptor internalization and Smad2/3 phosphorylation. TGFß1 stimulated Smad-dependent VEGF-A expression, VEGF receptor-mediated EC migration, and capillary-like tube formation, which were all abolished by either PI3K-C2α knockdown or a dynamin inhibitor. Finally, TGFß1-induced microvessel formation in Matrigel plugs was greatly attenuated in EC-specific PI3K-C2α-deleted mice. These observations indicate that PI3K-C2α plays the pivotal role in TGFß receptor endocytosis and thereby Smad2/3 signaling, participating in angiogenic actions of TGFß.
Asunto(s)
Endocitosis/genética , Péptidos y Proteínas de Señalización Intracelular/genética , Fosfatidilinositol 3-Quinasas/genética , Serina Endopeptidasas/genética , Factor de Crecimiento Transformador beta1/genética , Animales , Células Endoteliales/metabolismo , Regulación de la Expresión Génica , Células Endoteliales de la Vena Umbilical Humana , Humanos , Péptidos y Proteínas de Señalización Intracelular/biosíntesis , Ratones , Ratones Noqueados , Serina Endopeptidasas/biosíntesis , Transducción de Señal , Factor de Crecimiento Transformador beta1/metabolismo , Factor A de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
The phosphatidylinositol (PtdIns) 3-kinase (PI3K) family regulates diverse cellular processes, including cell proliferation, migration, and vesicular trafficking, through catalyzing 3'-phosphorylation of phosphoinositides. In contrast to class I PI3Ks, including p110α and p110ß, functional roles of class II PI3Ks, comprising PI3K-C2α, PI3K-C2ß, and PI3K-C2γ, are little understood. The lysophospholipid mediator sphingosine 1-phosphate (S1P) plays the important roles in regulating vascular functions, including vascular formation and barrier integrity, via the G-protein-coupled receptors S1P(1-3). We studied the roles of PI3K-C2α in S1P-induced endothelial cell (EC) migration and tube formation. S1P stimulated cell migration and activation of Akt, ERK, and Rac1, the latter of which acts as a signaling molecule essential for cell migration and tube formation, via S1P(1) in ECs. Knockdown of either PI3K-C2α or class I p110ß markedly inhibited S1P-induced migration, lamellipodium formation, and tube formation, whereas that of p110α or Vps34 did not. Only p110ß was necessary for S1P-iduced Akt activation, but both PI3K-C2α and p110ß were required for Rac1 activation. FRET imaging showed that S1P induced Rac1 activation in both the plasma membrane and PtdIns 3-phosphate (PtdIns(3)P)-enriched endosomes. Knockdown of PI3K-C2α but not p110ß markedly reduced PtdIns(3)P-enriched endosomes and suppressed endosomal Rac1 activation. Also, knockdown of PI3K-C2α but not p110ß suppressed S1P-induced S1P(1) internalization into PtdIns(3)P-enriched endosomes. Finally, pharmacological inhibition of endocytosis suppressed S1P-induced S1P(1) internalization, Rac1 activation, migration, and tube formation. These observations indicate that PI3K-C2α plays the crucial role in S1P(1) internalization into the intracellular vesicular compartment, Rac1 activation on endosomes, and thereby migration through regulating vesicular trafficking in ECs.
Asunto(s)
Fosfatidilinositol 3-Quinasas Clase II/fisiología , Regulación Enzimológica de la Expresión Génica , Receptores de Lisoesfingolípidos/genética , Movimiento Celular , Células Cultivadas , Fosfatidilinositol 3-Quinasas Clase II/genética , Endocitosis , Endosomas/metabolismo , Células Endoteliales/citología , Transferencia Resonante de Energía de Fluorescencia , Células Endoteliales de la Vena Umbilical Humana , Humanos , Lisofosfolípidos/metabolismo , ARN Interferente Pequeño/metabolismo , Transducción de Señal , Transfección , Proteínas de Unión al GTP rac/metabolismoRESUMEN
Fibrosis is a pathological process characterized by massive deposition of extracellular matrix (ECM) such as type I/III collagens and fibronectin that are secreted by an expanded pool of myofibroblasts, which are phenotypically altered fibroblasts with more contractile, proliferative, migratory and secretory activities. Fibrosis occurs in various organs including the lung, heart, liver and kidney, resulting in loss of normal tissue architecture and functions. Myofibroblasts could originate from multiple sources including tissue-resident fibroblasts, epithelial and endothelial cells through mechanisms of epithelial/endothelial-mesenchymal transition (EMT/EndMT), and bone marrow-derived circulating progenitors called fibrocytes. Emerging evidence in recent years shows that sphingosine-1-phosphate (S1P) acts on several types of target cells and is engaged in pro-fibrotic inflammatory process and fibrogenic process through multiple mechanisms, which include vascular permeability change, leukocyte infiltration, and migration, proliferation and myofibroblast differentiation of fibroblasts. Many of these S1P actions are receptor subtype-specific. In these actions, S1P has multiple cross-talks with other cytokines, particularly transforming growth factor-ß (TGFß), which plays a major role in fibrosis. The cross-talks include the regulation of S1P production through altered expression and activity of sphingosine kinases in fibrotic lesions, altered expression of S1P receptors, and S1P receptor-mediated transactivation of TGFß signaling pathway. These cross-talks may give rise to a feed-forward, amplifying loop between S1P and TGFß, and possibly with other cytokines in stimulating fibrogenesis. Another lysophospholipid mediator lysophosphatidic acid has also been recently implicated in fibrosis. The lysophospholipid signaling pathways represent novel, promising therapeutic targets for treating refractory fibrotic diseases. This article is part of a Special Issue entitled Advances in Lysophospholipid Research.
Asunto(s)
Progresión de la Enfermedad , Fibrosis/metabolismo , Fibrosis/patología , Lisofosfolípidos/metabolismo , Esfingosina/análogos & derivados , Animales , Humanos , Modelos Biológicos , Especificidad de Órganos , Esfingosina/metabolismoRESUMEN
BACKGROUND: Sphingosine-1-phosphate receptor 2 (S1P(2)) is expressed in vascular endothelial cells (ECs). However, the role of S1P(2) in vascular barrier integrity and anaphylaxis is not well understood. Endothelial nitric oxide synthase (eNOS) generates nitric oxide to mediate vascular leakage, compromising survival in patients with anaphylaxis. We recently observed that endothelial S1P(2) inhibits Akt, an activating kinase of eNOS. OBJECTIVE: We tested the hypothesis that endothelial S1P(2) might suppress eNOS, exerting a protective effect against endothelial barrier disruption and anaphylaxis. METHODS: Mice deficient in S1P(2) and eNOS underwent antigen challenge or platelet-activating factor (PAF) injection. Analyses were performed to examine vascular permeability and the underlying mechanisms. RESULTS: S1pr2 deletion augmented vascular leakage and lethality after either antigen challenge or PAF injection. PAF injection induced activation of Akt and eNOS in the aortas and lungs of S1pr2-null mice, which were augmented compared with values seen in wild-type mice. Consistently, PAF-induced increase in cyclic guanosine monophosphate levels in the aorta was enhanced in S1pr-null mice. Genetic Nos3 deletion or pharmacologic eNOS blockade protected S1pr2-null mice from aggravation of barrier disruption after antigen challenge and PAF injection. ECs isolated from S1pr2-null mice exhibited greater stimulation of Akt and eNOS, with enhanced nitric oxide production in response to sphingosine-1-phosphate or PAF, compared with that seen in wild-type ECs. Moreover, S1pr2-deficient ECs showed more severe disassembly of adherens junctions with augmented S-nitrosylation of ß-catenin in response to PAF, which was restored by pharmacologic eNOS blockade. CONCLUSION: S1P(2) diminishes harmful robust eNOS stimulation and thereby attenuates vascular barrier disruption, suggesting potential usefulness of S1P(2) agonists as novel therapeutic agents for anaphylaxis.
Asunto(s)
Anafilaxia/metabolismo , Óxido Nítrico Sintasa de Tipo III/antagonistas & inhibidores , Receptores de Lisoesfingolípidos/metabolismo , Uniones Adherentes/metabolismo , Anafilaxia/genética , Anafilaxia/mortalidad , Animales , Aorta/inmunología , Aorta/metabolismo , Permeabilidad Capilar/genética , Permeabilidad Capilar/inmunología , Modelos Animales de Enfermedad , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Activación Enzimática , Eliminación de Gen , Pulmón/inmunología , Pulmón/metabolismo , Ratones , Ratones Noqueados , NG-Nitroarginina Metil Éster/farmacología , Óxido Nítrico Sintasa de Tipo III/genética , Óxido Nítrico Sintasa de Tipo III/metabolismo , Factor de Activación Plaquetaria/farmacología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptores de Lisoesfingolípidos/genética , Transducción de Señal , beta Catenina/metabolismoRESUMEN
Phosphoinositide conversion regulates a diverse array of dynamic membrane events including endocytosis. However, it is not well understood which enzymes are involved in phosphoinositide conversions for receptor endocytosis. We found by small interfering RNA (siRNA)-mediated knockdown (KD) that class II PI3K α-isoform (PI3K-C2α), the 5'-phosphatase synaptojanin1 (Synj1), and the 4'-phosphatase INPP4B, but not PI3K-C2ß, Synj2, or INPP4A, were required for TGFß-induced endocytosis of TGFß receptor. TGFß induced rapid decreases in PI(4,5)P2 at the plasma membrane (PM) with increases in PI(4)P, followed by increases in PI(3,4)P2, in a TGFß receptor kinase ALK5-dependent manner. TGFß induced the recruitment of both synaptojanin1 and PI3K-C2α to the PM with their substantial colocalization. Knockdown of synaptojanin1 abolished TGFß-induced PI(4,5)P2 decreases and PI(4)P increases. Interestingly, PI3K-C2α KD abolished not only TGFß-induced PI(3,4)P2 increases but also TGFß-induced synaptojanin1 recruitment to the PM, PI(4,5)P2 decreases, and PI(4)P increases. Finally, the phosphoinositide conversions were necessary for TGFß-induced activation of Smad2 and Smad3. These observations demonstrate that the sequential phosphoinositide conversions mediated by Synj1, PI3K-C2α, and INPP4B are essential for TGFß receptor endocytosis and its signaling.
Asunto(s)
Endocitosis , Proteínas del Tejido Nervioso/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Fosfatidilinositoles/metabolismo , Monoéster Fosfórico Hidrolasas/metabolismo , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Transducción de Señal , Proteínas Smad/metabolismo , Receptores de Activinas Tipo II/metabolismo , Membrana Celular/metabolismo , Núcleo Celular/metabolismo , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Fosforilación , Receptor Tipo I de Factor de Crecimiento Transformador beta/metabolismo , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
Class II phosphatidylinositol 3-kinases (PI3K), PI3K-C2α and PI3K-C2ß, are involved in cellular processes including endocytosis, cilia formation and autophagy. However, the role of PI3K-C2α and PI3K-C2ß at the organismal level is not well understood. We found that double knockout (KO) mice with both smooth muscle-specific KO of PI3K-C2α and global PI3K-C2ß KO, but not single KO mice of either PI3K-C2α or PI3K-C2ß, exhibited reductions in arterial blood pressure and substantial attenuation of contractile responses of isolated aortic rings. In wild-type vascular smooth muscle cells, double knockdown of PI3K-C2α and PI3K-C2ß but not single knockdown of either PI3K markedly inhibited contraction with reduced phosphorylation of 20-kDa myosin light chain and MYPT1 and Rho activation, but without inhibition of the intracellular Ca2+ mobilization. These data indicate that PI3K-C2α and PI3K-C2ß play the redundant but essential role for vascular smooth muscle contraction and blood pressure regulation mainly through their involvement in Rho activation.
Asunto(s)
Calcio/metabolismo , Fosfatidilinositol 3-Quinasas Clase II/metabolismo , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/fisiología , Proteínas de Unión al GTP rho/metabolismo , Animales , Presión Sanguínea/fisiología , Células Cultivadas , Fosfatidilinositol 3-Quinasas Clase II/genética , Modelos Animales de Enfermedad , Isoenzimas , Ratones , Ratones Noqueados , Contracción Muscular/fisiología , Músculo Liso Vascular/citología , Músculo Liso Vascular/enzimología , Proteínas de Unión al GTP rho/genéticaRESUMEN
The plasma lysophospholipid mediator sphingosine-1-phosphate (S1P) is produced exclusively by sphingosine kinase (SPHK) 1 and SPHK2 in vivo, and plays diverse biological and pathophysiological roles by acting largely through three members of the G protein-coupled S1P receptors, S1P1, S1P2 and S1P3. S1P1 expressed on endothelial cells mediates embryonic vascular maturation and maintains vascular integrity by contributing to eNOS activation, inhibiting vascular permeability and inducing endothelial cell chemotaxis via Gi-coupled mechanisms. By contrast, S1P2, is expressed in high levels on vascular smooth muscle cells (VSMCs) and certain types of tumor cells, inhibiting Rac and cell migration via a G(12/13)-and Rho-dependent mechanism. In rat neointimal VSMCs, S1P1 is upregulated to mediate local production of platelet-derived growth factor, which is a key player in vascular remodeling. S1P3 expressed on endothelial cells also mediates chemotaxis toward S1P and vasorelaxation via NO production in certain vascular bed, playing protective roles for vascular integrity. S1P3 expressed on VSMCs and cardiac sinoatrial node cells mediates vasopressor and negative chronotropic effect, respectively. In addition, S1P3, together with S1P2 and SPHK1, is suggested to play a protective role against acute myocardial ischemia. However, our recent work indicates that overexpressed SPHK1 is involved in cardiomyocyte degeneration and fibrosis in vivo, in part through S1P activation of the S1P3 signaling. We also demonstrated that exogenously administered S1P accelerates neovascularization and blood flow recovery in ischemic limbs, suggesting its usefulness for angiogenic therapy. These results provide evidence for S1P receptor subtype-specific pharmacological intervention as a novel therapeutic approach to cardiovascular diseases and cancer.
Asunto(s)
Sistema Cardiovascular/metabolismo , Lisofosfolípidos/metabolismo , Transducción de Señal , Esfingosina/análogos & derivados , Animales , Humanos , Isquemia/metabolismo , Músculo Liso/metabolismo , Receptores de Lisoesfingolípidos/deficiencia , Receptores de Lisoesfingolípidos/genética , Receptores de Lisoesfingolípidos/metabolismo , Esfingosina/metabolismoRESUMEN
AIMS: The lysophospholipid mediator sphingosine-1-phosphate (S1P) acts on vascular endothelial cells to stimulate migration, proliferation, and capillary-like tube formation in vitro. It is unknown whether S1P stimulates in vivo angiogenesis induced under tissue ischaemia. We investigated the effects of both exogenously and endogenously overproduced S1P on post-ischaemic angiogenesis in murine hindlimbs. METHODS AND RESULTS: The effects of locally injected S1P on blood flow recovery, angiogenesis, and vascular permeability in mouse ischaemic hindlimbs that underwent femoral arteriectomy were assessed by a laser Doppler blood flow (LDBF) analysis, anti-CD31 immunohistochemistry, and Miles assay, respectively, and compared with those induced by fibroblast growth factor (FGF)-2. Blood flow recovery and angiogenesis in sphingosine kinase 1-transgenic mice that overproduce S1P endogenously were also assessed and compared with wild-type mice. The LDBF analysis showed that daily intramuscular administration of S1P dose-dependently stimulated blood flow recovery, resulting in up to twice as much blood flow when compared with vehicle control, which was accompanied by 1.7-fold increase in the capillary density. The optimal S1P effects were comparable with those obtained with FGF-2. S1P injection did not increase vascular permeability. The post-ischaemic blood flow recovery and angiogenesis were accelerated in sphingosine kinase 1-transgenic mice, which showed 40-fold higher sphingosine kinase activity and 1.8-fold higher S1P content in skeletal muscle than in wild-type (WT) mice, without an increase in the vascular permeability when compared with WT mice. CONCLUSION: These results indicate that either local exogenous S1P administration or endogenous S1P overproduction promotes post-ischaemic angiogenesis and blood flow recovery. These observations suggest potential therapeutic usefulness of S1P for tissue ischaemia.
Asunto(s)
Inductores de la Angiogénesis/metabolismo , Isquemia/metabolismo , Lisofosfolípidos/metabolismo , Músculo Esquelético/irrigación sanguínea , Neovascularización Fisiológica , Esfingosina/análogos & derivados , Animales , Velocidad del Flujo Sanguíneo , Capilares/metabolismo , Capilares/fisiopatología , Permeabilidad Capilar , Modelos Animales de Enfermedad , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Miembro Posterior , Inmunohistoquímica , Isquemia/fisiopatología , Flujometría por Láser-Doppler , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/metabolismo , Flujo Sanguíneo Regional , Esfingosina/metabolismo , Factores de TiempoRESUMEN
AIMS: The lysophospholipid mediator sphingosine-1-phosphate (S1P) activates G protein-coupled receptors (GPCRs) to induce potent inhibition of platelet-derived growth factor (PDGF)-induced Rac activation and, thereby, chemotaxis in rat vascular smooth muscle cells (VSMCs). We explored the heterotrimeric G protein and the downstream mechanism that mediated S1P inhibition of Rac and cell migration in VSMCs. METHODS AND RESULTS: S1P inhibition of PDGF-induced cell migration and Rac activation in VSMCs was abolished by the selective S1P(2) receptor antagonist JTE-013. The C-terminal peptides of Galpha subunits (Galpha-CTs) act as specific inhibitors of respective G protein-GPCR coupling. Adenovirus-mediated expression of Galpha(12)-CT, Galpha(13)-CT, and Galpha(q)-CT, but not that of Galpha(s)-CT or LacZ or pertussis toxin treatment, abrogated S1P inhibition of PDGF-induced Rac activation and migration, indicating that both G(12/13) and G(q) classes are necessary for the S1P inhibition. The expression of Galpha(q)-CT as well as Galpha(12)-CT and Galpha(13)-CT also abolished S1P-induced Rho stimulation. C3 toxin, but not a Rho kinase inhibitor or a dominant negative form of Rho kinase, abolished S1P inhibition of PDGF-induced Rac activation and cell migration. The angiotensin II receptor AT(1), which robustly couples to G(q), did not mediate either Rho activation or inhibition of PDGF-induced Rac activation or migration, suggesting that activation of G(q) alone was not sufficient for Rho activation and resultant Rac inhibition. However, the AT(1) receptor fused to Galpha(12) was able to induce not only Rho stimulation but also inhibition of PDGF-induced Rac activation and migration. Phospholipase C inhibition did not affect S1P-induced Rho activation, and protein kinase C activation by a phorbol ester did not mimic S1P action, suggesting that S1P inhibition of migration or Rac was not dependent on the phospholipase C pathway. CONCLUSION: These observations together suggest that S1P(2) mediates inhibition of Rac and migration through the coordinated action of G(12/13) and G(q) for Rho activation in VSMCs.
Asunto(s)
Movimiento Celular , Subunidades alfa de la Proteína de Unión al GTP G12-G13/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/metabolismo , Lisofosfolípidos/metabolismo , Músculo Liso Vascular/enzimología , Receptores de Lisoesfingolípidos/metabolismo , Esfingosina/análogos & derivados , Proteínas de Unión al GTP rac/metabolismo , Proteínas de Unión al GTP rho/metabolismo , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/análogos & derivados , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/farmacología , ADP Ribosa Transferasas/farmacología , Animales , Toxinas Botulínicas/farmacología , Calcio/metabolismo , Movimiento Celular/efectos de los fármacos , Células Cultivadas , Relación Dosis-Respuesta a Droga , Subunidades alfa de la Proteína de Unión al GTP G12-G13/genética , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/genética , Masculino , Músculo Liso Vascular/efectos de los fármacos , Proteína Quinasa C/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-sis/metabolismo , Pirazoles/farmacología , Piridinas/farmacología , Ratas , Ratas Wistar , Receptor de Angiotensina Tipo 1/genética , Receptor de Angiotensina Tipo 1/metabolismo , Receptores de Lisoesfingolípidos/antagonistas & inhibidores , Proteínas Recombinantes de Fusión/metabolismo , Transducción de Señal , Esfingosina/metabolismo , Transfección , Proteínas de Unión al GTP rac/antagonistas & inhibidores , Quinasas Asociadas a rho/antagonistas & inhibidores , Quinasas Asociadas a rho/metabolismoRESUMEN
Pinocytosis is an important fundamental cellular process that is used by the cell to transport fluid and solutes. Phosphoinositide 3-kinases (PI3Ks) regulate a diverse array of dynamic membrane events. However, it is not well-understood which PI3K isoforms are involved in specific mechanisms of pinocytosis. We performed knockdown studies of endogenous PI3K isoforms and clathrin heavy chain (CHC) mediated by small interfering RNA (siRNA). The results demonstrated that the class II PI3K PI3K-C2α and PI3K-C2ß, but not the class I or III PI3K, were required for pinocytosis, based on an evaluation of fluorescein-5-isothiocyanate (FITC)-dextran uptake in endothelial cells. Pinocytosis was partially dependent on both clathrin and dynamin, and both PI3K-C2α and PI3K-C2ß were required for clathrin-mediated-but not clathrin-non-mediated-FITC-dextran uptake at the step leading up to its delivery to early endosomes. Both PI3K-C2α and PI3K-C2ß were co-localized with clathrin-coated pits and vesicles. However, PI3K-C2ß, but not PI3K-C2α, was highly co-localized with actin filament-associated clathrin-coated structures and required for actin filament formation at the clathrin-coated structures. These results indicate that PI3K-C2α and PI3K-C2ß play differential, indispensable roles in clathrin-mediated pinocytosis.
Asunto(s)
Fosfatidilinositol 3-Quinasas Clase II/metabolismo , Clatrina/metabolismo , Células Endoteliales/metabolismo , Células Endoteliales/fisiología , Fosfatidilinositol 3-Quinasas/metabolismo , Pinocitosis/fisiología , Citoesqueleto de Actina/metabolismo , Animales , Células Cultivadas , Células Endoteliales de la Vena Umbilical Humana , Humanos , ARN Interferente Pequeño/metabolismoRESUMEN
Class II phosphoinositide 3-kinases (PI3Ks), PI3K-C2α and PI3K-C2ß, are highly homologous and distinct from class I and class III PI3Ks in catalytic products and domain structures. In contrast to class I and class III PI3Ks, physiological roles of PI3K-C2α and PI3K-C2ß are not fully understood. Because we previously demonstrated that PI3K-C2α is involved in vascular smooth muscle contraction, we studied the phenotypes of smooth muscle-specific knockout (KO) mice of PI3K-C2α and PI3K-C2ß. The pup numbers born from single PI3K-C2α-KO and single PI3K-C2ß-KO mothers were similar to those of control mothers, but those from double KO (DKO) mothers were smaller compared with control mice. However, the number of intrauterine fetuses in pregnant DKO mothers was similar to that in control mice. Both spontaneous and oxytocin-induced contraction of isolated uterine smooth muscle (USM) strips was diminished in DKO mice but not in either of the single KO mice, compared with control mice. Furthermore, contraction of USM of DKO mice was less sensitive to a Rho kinase inhibitor. Mechanistically, the extent of oxytocin-induced myosin light chain phosphorylation was greatly reduced in USM from DKO mice compared with control mice. The oxytocin-induced rise in the intracellular Ca2+ concentration in USM was similar in DKO and control mice. However, Rho activation in the intracellular compartment was substantially attenuated in DKO mice compared with control mice, as evaluated by fluorescence resonance energy transfer imaging technique. These data indicate that both PI3K-C2α and PI3K-C2ß are required for normal USM contraction and parturition mainly through their involvement in Rho activation.
Asunto(s)
Fosfatidilinositol 3-Quinasas Clase II/metabolismo , Músculo Liso Vascular/enzimología , Parto , Fosfatidilinositol 3-Quinasas/metabolismo , Contracción Uterina , Útero/enzimología , Proteína de Unión al GTP rhoA/metabolismo , Animales , Fosfatidilinositol 3-Quinasas Clase II/genética , Femenino , Ratones , Ratones Noqueados , Contracción Muscular , Músculo Liso Vascular/fisiología , Cadenas Ligeras de Miosina , Fosfatidilinositol 3-Quinasas/genética , Fosforilación , Útero/fisiología , Proteína de Unión al GTP rhoA/genéticaRESUMEN
Atherosclerosis is the major cause of ischemic coronary heart diseases and characterized by the infiltration of cholesterol-accumulating macrophages in the vascular wall. Although sphingolipids are implicated in atherosclerosis as both membrane components and lipid mediators, the precise role of sphingolipids in atherosclerosis remains elusive. Here, we found that genetic deficiency of sphingosine kinase-2 (SphK2) but not SphK1 aggravates the formation of atherosclerotic lesions in mice with ApoE deficiency. Bone marrow chimaera experiments show the involvement of SphK2 expressed in bone marrow-derived cells. In macrophages, deficiency of SphK2, a major SphK isoform in this cell type, results in increases in cellular sphingosine and ceramides. SphK2-deficient macrophages have increases in lipid droplet-containing autophagosomes and autolysosomes and defective lysosomal degradation of lipid droplets via autophagy with an impaired luminal acidic environment and proteolytic activity in the lysosomes. Transgenic overexpression of SphK1 in SphK2-deficient mice rescued aggravation of atherosclerosis and abnormalities of autophagosomes and lysosomes in macrophages with reductions of sphingosine, suggesting at least partial overlapping actions of two SphKs. Taken together, these results indicate that SphK2 is required for autophagosome- and lysosome-mediated catabolism of intracellular lipid droplets to impede the development of atherosclerosis; therefore, SphK2 may be a novel target for treating atherosclerosis.
Asunto(s)
Apolipoproteínas E/genética , Aterosclerosis/genética , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Animales , Aterosclerosis/metabolismo , Aterosclerosis/patología , Células de la Médula Ósea/metabolismo , Colesterol/metabolismo , Modelos Animales de Enfermedad , Humanos , Metabolismo de los Lípidos/genética , Lípidos/genética , Macrófagos/metabolismo , Macrófagos/patología , Ratones , Esfingolípidos/genética , Esfingolípidos/metabolismo , Esfingosina/metabolismoRESUMEN
We report a case of mixed epithelial and stromal tumor of the kidney (MESTK) in a 32-week-old heterozygous sphingosine 1-phosphate-2 (S1P2) receptor deficient female mouse. A white solid mass replacing the left kidney was observed at the left retroperitoneal wall. Histologically, the tumor mass consisted of dimorphic cellular components of epithelial and stromal cells. Epithelial cells formed various sized irregular-shaped tubular structures resembling renal tubules surrounded by stromal cells. Immunohistochemically, epithelial cells were positive for cytokeratin, while stromal cells showed positive immunoreactivity with alpha-smooth muscle actin as well as vimentin. Based on the morphological and immunohistochemical findings, this tumor was diagnosed as a MESTK.
Asunto(s)
Neoplasias Renales/clasificación , Neoplasias Glandulares y Epiteliales/patología , Receptores de Lisoesfingolípidos/genética , Animales , Etilnitrosourea , Femenino , Eliminación de Gen , Heterocigoto , Neoplasias Renales/patología , RatonesRESUMEN
Idiopathic pulmonary fibrosis is a devastating disease with poor prognosis. The pathogenic role of the lysophospholipid mediator sphingosine-1-phosphate and its receptor S1PR2 in lung fibrosis is unknown. We show here that genetic deletion of S1pr2 strikingly attenuated lung fibrosis induced by repeated injections of bleomycin in mice. We observed by using S1pr2LacZ/+ mice that S1PR2 was expressed in alveolar macrophages, vascular endothelial cells and alveolar epithelial cells in the lung and that S1PR2-expressing cells accumulated in the fibrotic legions. Bone marrow chimera experiments suggested that S1PR2 in bone marrow-derived cells contributes to the development of lung fibrosis. Depletion of macrophages greatly attenuated lung fibrosis. Bleomycin administration stimulated the mRNA expression of the profibrotic cytokines IL-13 and IL-4 and the M2 markers including arginase 1, Fizz1/Retnla, Ccl17 and Ccl24 in cells collected from broncho-alveolar lavage fluids (BALF), and S1pr2 deletion markedly diminished the stimulated expression of these genes. BALF cells from bleomycin-administered wild-type mice showed a marked increase in phosphorylation of STAT6, a transcription factor which is activated downstream of IL-13, compared with saline-administered wild-type mice. Interestingly, in bleomycin-administered S1pr2-/- mice, STAT6 phosphorylation in BALF cells was substantially diminished compared with wild-type mice. Finally, pharmacological S1PR2 blockade in S1pr2+/+ mice alleviated bleomycin-induced lung fibrosis. Thus, S1PR2 facilitates lung fibrosis through the mechanisms involving augmentation of IL-13 expression and its signaling in BALF cells, and represents a novel target for treating lung fibrosis.
Asunto(s)
Fibrosis Pulmonar Idiopática/etiología , Interleucina-13/metabolismo , Macrófagos/metabolismo , Receptores de Lisoesfingolípidos/metabolismo , Animales , Bleomicina/toxicidad , Líquido del Lavado Bronquioalveolar/química , Líquido del Lavado Bronquioalveolar/citología , Modelos Animales de Enfermedad , Fibrosis Pulmonar Idiopática/metabolismo , Fibrosis Pulmonar Idiopática/patología , Interleucina-13/genética , Macrófagos/efectos de los fármacos , Macrófagos/patología , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores de Lisoesfingolípidos/deficiencia , Receptores de Lisoesfingolípidos/genética , Factor de Transcripción STAT6/metabolismo , Transducción de Señal , Receptores de Esfingosina-1-Fosfato , Quimera por Trasplante/genética , Quimera por Trasplante/metabolismo , Regulación hacia ArribaRESUMEN
The G protein-coupled receptors S1P2/Edg5 and S1P3/Edg3 both mediate sphingosine-1-phosphate (S1P) stimulation of Rho, yet S1P2 but not S1P3 mediates downregulation of Rac activation, membrane ruffling, and cell migration in response to chemoattractants. Specific inhibition of endogenous Galpha12 and Galpha13, but not of Galphaq, by expression of respective C-terminal peptides abolished S1P2-mediated inhibition of Rac, membrane ruffling, and migration, as well as stimulation of Rho and stress fiber formation. Fusion receptors comprising S1P2 and either Galpha12 or Galpha13, but not Galphaq, mediated S1P stimulation of Rho and also inhibition of Rac and migration. Overexpression of Galphai, by contrast, specifically antagonized S1P2-mediated inhibition of Rac and migration. The S1P2 actions were mimicked by expression of V14Rho and were abolished by C3 toxin and N19Rho, but not Rho kinase inhibitors. In contrast to S1P2, S1P3 mediated S1P-directed, pertussis toxin-sensitive chemotaxis and Rac activation despite concurrent stimulation of Rho via G12/13. Upon inactivation of Gi by pertussis toxin, S1P3 mediated inhibition of Rac and migration just like S1P2. These results indicate that integration of counteracting signals from the Gi- and the G12/13-Rho pathways directs either positive or negative regulation of Rac, and thus cell migration, upon activation of a single S1P receptor isoform.
Asunto(s)
Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Proteínas de Unión al GTP Heterotriméricas/metabolismo , Receptores de Superficie Celular/química , Receptores Acoplados a Proteínas G , Proteína de Unión al GTP rac1/metabolismo , Células 3T3 , Adenoviridae/genética , Adenoviridae/metabolismo , Animales , Western Blotting , Toxinas Botulínicas/metabolismo , Células CHO , Células COS , Movimiento Celular , Quimiotaxis , Cricetinae , Medio de Cultivo Libre de Suero/farmacología , Relación Dosis-Respuesta a Droga , Subunidades alfa de la Proteína de Unión al GTP G12-G13 , Ratones , Microscopía Fluorescente , Péptidos/química , Toxina del Pertussis/farmacología , Plásmidos/metabolismo , Isoformas de Proteínas , Receptores de Superficie Celular/metabolismo , Receptores Lisofosfolípidos , Proteínas Recombinantes de Fusión/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal , Factores de Tiempo , Transfección , Proteínas de Unión al GTP rac/metabolismo , Proteínas de Unión al GTP rho/metabolismoRESUMEN
We demonstrated previously that membrane depolarization and excitatory receptor agonists such as noradrenaline induce Ca2+-dependent Rho activation in VSM (vascular smooth muscle), resulting in MP (myosin phosphatase) inhibition through the mechanisms involving Rho kinase-mediated phosphorylation of its regulatory subunit MYPT1. In the present study, we show in de-endothelialized VSM strips that the PI3K (phosphoinositide 3-kinase) inhibitors LY294002 and wortmannin inhibited KCl membrane depolarization- and noradrenaline-induced Rho activation and MYPT1 phosphorylation, with concomitant inhibition of MLC (20-kDa myosin light chain) phosphorylation and contraction. LY294002 also augmented de-phosphorylation of MLC and resultantly relaxation in KCl-contracted VSM, whereas LY294002 was much less effective or ineffective under the conditions in which MP was inhibited by either a phosphatase inhibitor or a phorbol ester in Rho-independent manners. VSM express at least four PI3K isoforms, including the class I enzymes p110alpha and p110beta and the class II enzymes PI3K-C2alpha and -C2beta. The dose-response relationships of PI3K-inhibitor-induced inhibition of Rho, MLC phosphorylation and contraction were similar to that of PI3K-C2alpha inhibition, but not to that of the class I PI3K inhibition. Moreover, KCl and noradrenaline induced stimulation of PI3K-C2alpha in a Ca2+-dependent manner, but not of p110alpha or p110beta. Down-regulation of PI3K-C2alpha expression by siRNA (small interfering RNA) inhibited contraction and phosphorylation of MYPT1 and MLC in VSM cells. Finally, intravenous wortmannin infusion induced sustained hypotension in rats, with inhibition of PI3K-C2alpha activity, GTP-loading of Rho and MYPT1 phosphorylation in the artery. These results indicate the novel role of PI3K-C2alpha in Ca2+-dependent Rho-mediated negative control of MP and thus VSM contraction.
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Contracción Muscular/fisiología , Músculo Liso Vascular/enzimología , Músculo Liso Vascular/fisiología , Fosfatasa de Miosina de Cadena Ligera/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas de Unión al GTP rho/metabolismo , Animales , Aorta/metabolismo , Calcio/metabolismo , Fosfatidilinositol 3-Quinasas Clase II , Regulación hacia Abajo , Regulación Enzimológica de la Expresión Génica , Masculino , Músculo Liso Vascular/metabolismo , Fosfatidilinositol 3-Quinasas/química , Inhibidores de las Quinasa Fosfoinosítidos-3 , Fosforilación , Interferencia de ARN , ARN Interferente Pequeño , Conejos , Ratas , Ratas WistarRESUMEN
BACKGROUND: Cardiac fibroblasts, together with cardiomyocytes, occupy the majority of cells in the myocardium and are involved in myocardial remodeling. The lysophospholipid mediator sphigosine-1-phosphate (S1P) regulates functions of cardiovascular cells through multiple receptors including S1PR1-S1PR3. S1PR1 but not other S1P receptors was upregulated in angiotensin II-induced hypertrophic hearts. Therefore, we investigated a role of S1PR1 in fibroblasts for cardiac remodeling by employing transgenic mice that overexpressed S1PR1 under the control of α-smooth muscle actin promoter. In S1PR1-transgenic mouse heart, fibroblasts and/or myofibroblasts were hyperplastic, and those cells as well as vascular smooth muscle cells overexpressed S1PR1. Transgenic mice developed bi-ventricular hypertrophy by 12-week-old and diffuse interstitial fibrosis by 24-week-old without hemodynamic stress. Cardiac remodeling in transgenic mice was associated with greater ERK phosphorylation, upregulation of fetal genes, and systolic dysfunction. Transgenic mouse heart showed increased mRNA expression of angiotensin-converting enzyme and interleukin-6 (IL-6). Isolated fibroblasts from transgenic mice exhibited enhanced generation of angiotensin II, which in turn stimulated IL-6 release. Either an AT1 blocker or angiotensin-converting enzyme inhibitor prevented development of cardiac hypertrophy and fibrosis, systolic dysfunction and increased IL-6 expression in transgenic mice. Finally, administration of anti-IL-6 antibody abolished an increase in tyrosine phosphorylation of STAT3, a major signaling molecule downstream of IL-6, in the transgenic mouse heart and prevented development of cardiac hypertrophy in transgenic mice. These results demonstrate a promoting role of S1PR1 in cardiac fibroblasts for cardiac remodeling, in which angiotensin II-AT1 and IL-6 are involved.
Asunto(s)
Angiotensina II/metabolismo , Interleucina-6/metabolismo , Receptores de Lisoesfingolípidos/metabolismo , Transducción de Señal , Angiotensina II/análisis , Inhibidores de la Enzima Convertidora de Angiotensina/farmacología , Inhibidores de la Enzima Convertidora de Angiotensina/uso terapéutico , Animales , Bencimidazoles/farmacología , Bencimidazoles/uso terapéutico , Compuestos de Bifenilo , Cardiomegalia/diagnóstico por imagen , Cardiomegalia/patología , Cardiomegalia/prevención & control , Células Cultivadas , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Ventrículos Cardíacos/diagnóstico por imagen , Humanos , Interleucina-6/análisis , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Miocardio/metabolismo , Miocardio/patología , Miocitos Cardíacos/citología , Miocitos Cardíacos/metabolismo , Peptidil-Dipeptidasa A/genética , Peptidil-Dipeptidasa A/metabolismo , Fosforilación/efectos de los fármacos , Plásmidos/genética , Plásmidos/metabolismo , Receptores de Lisoesfingolípidos/genética , Transducción de Señal/efectos de los fármacos , Tetrazoles/farmacología , Tetrazoles/uso terapéuticoRESUMEN
Ca2+ sensitization of vascular smooth muscle (VSM) contraction involves Rho-dependent and Rho-kinase-dependent suppression of myosin phosphatase activity. We previously demonstrated that excitatory agonists in fact induce activation of RhoA in VSM. In this study, we demonstrate a novel Ca2+-dependent mechanism for activating RhoA in rabbit aortic VSM. High KCl-induced membrane depolarization as well as noradrenalin stimulation induced similar extents of sustained contraction in rabbit VSM. Both stimuli also induced similar extents of time-dependent, sustained increases in the amount of an active GTP-bound form of RhoA. Consistent with this, the Rho kinase inhibitors HA1077 and Y27632 inhibited both contraction and the 20-kDa myosin light chain phosphorylation induced by KCl as well as noradrenalin, with similar dose-response relations. Either removal of extracellular Ca2+ or the addition of a dihydropyridine Ca2+ channel antagonist totally abolished KCl-induced Rho stimulation and contraction. The calmodulin inhibitor W7 suppressed KCl-induced Rho activation and contraction. Ionomycin mimicked W7-sensitive Rho activation. The expression of dominant-negative N19RhoA suppressed Ca2+-induced Thr695 phosphorylation of the 110-kDa regulatory subunit of myosin phosphatase and phosphorylation of myosin light chain in VSM cells. Finally, either the combination of extracellular Ca2+ removal and depletion of the intracellular Ca2+ store or the addition of W7 greatly reduced noradrenalin-induced and the thromboxane A2 analogue-induced Rho stimulation and contraction. Taken together, these results indicate the existence of the thus-far unrecognized Ca2+-dependent Rho stimulation mechanism in VSM. Excitatory receptor agonists are suggested to use this pathway for simulating Rho.