RESUMEN
Disordered expression and distribution of plasma membrane proteins at the cell surface leads to diverse malignant phenotypes in tumors, including cell invasion. The ubiquitin-specific protease TRE17/USP6, an oncogene identified in Ewing sarcoma, is highly expressed in several cancers and locally aggressive tumor-like lesions. We have previously demonstrated that TRE17 regulates the trafficking of plasma membrane proteins that enter cells via clathrin-independent endocytosis (CIE); TRE17 prevents CIE cargo proteins from being targeted to lysosomes for degradation by deubiquitylating them. However, functional insights into the effects of TRE17-mediated CIE cargo trafficking on cell invasion remain unknown. Here, we show that increased expression of TRE17 enhances invasiveness of the human sarcoma cell line HT-1080 by elevating the cell surface levels of the membrane glycoprotein CD147, which plays a central role in tumor progression. We demonstrate overexpression of TRE17 decreases ubiquitylated CD147, which is accompanied by suppression of CD147 transport to lysosomes, resulting in the stabilization and increase of cell surface-localized CD147. On the other hand, we show knockdown of TRE17 decreases cell surface CD147, which is coupled with reduced production of matrix metalloproteinases, the enzymes responsible for extracellular matrix degradation. Furthermore, we demonstrate that inhibition of CD147 by a specific inhibitor alleviated the TRE17-promoted tumor cell invasion. We therefore propose a model for the pathogenesis of TRE17-driven tumors in which TRE17 increases CD147 at the cell surface by preventing its lysosomal degradation, which in turn enhances matrix metalloproteinase synthesis and matrix degradation, thereby promoting tumor cell invasion.
Asunto(s)
Basigina , Neoplasias Óseas , Proteínas de la Membrana , Sarcoma de Ewing , Ubiquitina Tiolesterasa , Proteasas Ubiquitina-Específicas , Basigina/metabolismo , Neoplasias Óseas/enzimología , Neoplasias Óseas/patología , Línea Celular Tumoral , Clatrina/genética , Humanos , Metaloproteinasas de la Matriz/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Invasividad Neoplásica , Sarcoma de Ewing/enzimología , Sarcoma de Ewing/patología , Ubiquitina Tiolesterasa/genética , Ubiquitina Tiolesterasa/metabolismo , Proteasas Ubiquitina-Específicas/genética , Proteasas Ubiquitina-Específicas/metabolismoRESUMEN
ADP-ribosylation factor (Arf) family consisting of six family members, Arf1-Arf6, belongs to Ras superfamily and orchestrates vesicle trafficking under the control of guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins. It is well established that brefeldin A, a potent inhibitor of ArfGEFs, blocks cytokine secretion from activated T cells, suggesting that the Arf pathway plays important roles in T cell functions. In this study, because Arf1 and Arf6 are the best-characterized members among Arf family, we established T lineage-specific Arf1-deficient, Arf6-deficient, and Arf1/6 double-deficient mice to understand physiological roles of the Arf pathway in the immune system. Contrary to our expectation, Arf deficiency had little or no impact on cytokine secretion from the activated T cells. In contrast, the lack of both Arf1 and Arf6, but neither Arf1 nor Arf6 deficiency alone, rendered naive T cells susceptible to apoptosis upon TCR stimulation because of imbalanced expression of Bcl-2 family members. We further demonstrate that Arf1/6 deficiency in T cells alleviates autoimmune diseases like colitis and experimental autoimmune encephalomyelitis, whereas Ab response under Th2-polarizing conditions is seemingly normal. Our findings reveal an unexpected role for the Arf pathway in the survival of T cells during TCR-induced activation and its potential as a therapeutic target in the autoimmune diseases.
Asunto(s)
Factor 1 de Ribosilacion-ADP/metabolismo , Factores de Ribosilacion-ADP/metabolismo , Colitis/inmunología , Encefalomielitis Autoinmune Experimental/inmunología , Linfocitos T/inmunología , Factor 1 de Ribosilacion-ADP/genética , Factor 6 de Ribosilación del ADP , Factores de Ribosilacion-ADP/genética , Animales , Apoptosis , Supervivencia Celular , Células Cultivadas , Inmunoterapia , Activación de Linfocitos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Receptores de Antígenos de Linfocitos T/metabolismo , Transducción de SeñalRESUMEN
A switch in the response of commissural axons to the repellent Slit is crucial for ensuring that they cross the ventral midline only once. However, the underlying mechanisms remain to be elucidated. We have found that both endocytosis and recycling of Robo1 receptor are crucial for modulating Slit sensitivity in vertebrate commissural axons. Robo1 endocytosis and its recycling back to the cell surface maintained the stability of axonal Robo1 during Slit stimulation. We identified Arf6 guanosine triphosphatase and its activators, cytohesins, as previously unknown components in Slit-Robo1 signalling in vertebrate commissural neurons. Slit-Robo1 signalling activated Arf6. The Arf6-deficient mice exhibited marked defects in commissural axon midline crossing. Our data showed that a Robo1 endocytosis-triggered and Arf6-mediated positive-feedback strengthens the Slit response in commissural axons upon their midline crossing. Furthermore, the cytohesin-Arf6 pathways modulated this self-enhancement of the Slit response before and after midline crossing, resulting in a switch that reinforced robust regulation of axon midline crossing. Our study provides insights into endocytic trafficking-mediated mechanisms for spatiotemporally controlled axonal responses and uncovers new players in the midline switch in Slit responsiveness of commissural axons.
Asunto(s)
Factores de Ribosilacion-ADP/metabolismo , Axones/metabolismo , Endocitosis/fisiología , Proteínas del Tejido Nervioso/metabolismo , Receptores Inmunológicos/metabolismo , Transducción de Señal/fisiología , Factor 6 de Ribosilación del ADP , Factores de Ribosilacion-ADP/genética , Animales , Células HEK293 , Humanos , Ratones , Ratones Noqueados , Proteínas RoundaboutRESUMEN
Many plasma membrane proteins enter cells by clathrin-independent endocytosis (CIE). Rab family small GTPases play pivotal roles in CIE and following intracellular trafficking of cargo proteins. Here, we provide evidence that TBC1D24, which contains an atypical Rab GAP domain, facilitates formation of tubular recycling endosomes (TREs) that are a hallmark of the CIE cargo trafficking pathway in HeLa cells. Overexpression of TBC1D24 in HeLa cells dramatically increased TREs loaded with CIE cargo proteins, while deletion of TBC1D24 impaired TRE formation and delayed the recycling of CIE cargo proteins back to the plasma membrane. We also found that TBC1D24 binds to Rab22A, through which TBC1D24 regulates TRE-mediated CIE cargo recycling. These findings provide insight into regulatory mechanisms for CIE cargo trafficking.
Asunto(s)
Clatrina/metabolismo , Endocitosis , Endosomas/metabolismo , Proteínas Activadoras de GTPasa/metabolismo , Proliferación Celular , Proteína-1 Reguladora de Fusión/metabolismo , Células HEK293 , Células HeLa , Humanos , Transporte de Proteínas , Proteínas de Unión al GTP rab/metabolismoRESUMEN
Chemoattractant sensing, adhesiveness, and migration are critical events underlying the recruitment of neutrophils (PMNs) to sites of inflammation or infection. Defects in leukocyte adhesion or migration result in immunodeficiency disorders characterized by recurrent infections. In this study, we evaluated the role of Arf6 on PMN adhesion in vitro and on migration to inflammatory sites using PMN-Arf6 conditional knockout (cKO) mice. In PMN-like PLB-985 silenced for Arf6 fMLP-mediated adhesion to the ß2 integrin ligands, ICAM-1 and fibrinogen or the ß1/ß2 integrin ligand fibronectin was significantly reduced. Furthermore, overexpression of wild-type Arf6 promoted basal and fMLP-induced adhesion to immobilized integrin ligands, while overexpression of the dominant-negative Arf6 has the opposite effects. Using the Elane-Cre deleting mouse strains, we report that the level of Arf6 deletion in inflammatory PMNs isolated from the dorsal air pouches was stronger when compared to naïve cells isolated from the bone marrow. In PMN-Arf6 cKO mice, the recruitment of PMNs into the dorsal air pouch injected with LPS or the chemoattractant fMLP was significantly diminished. Impaired cell migration correlated with reduced cell surface expression of CD11a and CD11b in Arf6 cKO PMNs. Our results highlight that Arf6 regulates the activity and possibly the recycling of PMN integrins, and this compromises PMN migration to inflammatory sites.
Asunto(s)
Neutrófilos/citología , Neutrófilos/metabolismo , Factor 6 de Ribosilación del ADP , Factores de Ribosilacion-ADP/genética , Factores de Ribosilacion-ADP/metabolismo , Animales , Apoptosis/genética , Apoptosis/fisiología , Recuento de Células Sanguíneas , Western Blotting , Línea Celular , Células Cultivadas , Proteínas de la Matriz Extracelular/genética , Proteínas de la Matriz Extracelular/metabolismo , Citometría de Flujo , Vectores Genéticos/genética , Humanos , Cadenas beta de Integrinas/genética , Cadenas beta de Integrinas/metabolismo , Antígeno-1 Asociado a Función de Linfocito/genética , Antígeno-1 Asociado a Función de Linfocito/metabolismo , Antígeno de Macrófago-1/genética , Antígeno de Macrófago-1/metabolismo , Ratones , Ratones Noqueados , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
A hallmark of clear cell renal cell carcinoma (ccRCC) is the presence of intracellular lipid droplets (LD) and it is assumed that phosphatidic acid (PA) produced by phospholipase D (PLD) plays some role in the LD formation. However, little is known about the significance of PLD in ccRCC. In this study, we examined the expression levels of PLD in ccRCC. The classical mammalian isoforms of PLD are PLD1 and PLD2, and the levels of both mRNA were higher at the primary tumor sites than in normal kidney tissues. Similarly, both PLD were significantly abundant in tumor cells as determined by analysis using immunohistochemical staining. Importantly, a higher level of PLD was significantly associated with a higher tumor stage and grade. Because PLD2 knockdown effectively suppressed the cell proliferation and invasion of ccRCC as compared with PLD1 in vitro, we examined the effect of PLD2 in vivo. Notably, shRNA-mediated knockdown of PLD2 suppressed the growth and invasion of tumors in nude mouse xenograft models. Moreover, the higher expression of PLD2 was significantly associated with poorer prognosis in 67 patients. As for genes relating to the tumor invasion of PLD2, we found that angiogenin (ANG) was positively regulated by PLD2. In fact, the expression levels of ANG were elevated in tumor tissues as compared with normal kidney and the inhibition of ANG activity with a neutralizing antibody significantly suppressed tumor invasion. Overall, we revealed for the first time that PLD2-produced PA promoted cell invasion through the expression of ANG in ccRCC cells.
Asunto(s)
Carcinoma de Células Renales/genética , Regulación Neoplásica de la Expresión Génica , Neoplasias Renales/genética , Fosfolipasa D/genética , Ribonucleasa Pancreática/genética , Anciano , Animales , Carcinoma de Células Renales/metabolismo , Carcinoma de Células Renales/patología , Línea Celular Tumoral , Progresión de la Enfermedad , Células HEK293 , Humanos , Estimación de Kaplan-Meier , Neoplasias Renales/metabolismo , Neoplasias Renales/patología , Ratones Desnudos , Persona de Mediana Edad , Fosfolipasa D/metabolismo , Interferencia de ARN , Tratamiento con ARN de Interferencia/métodos , Ribonucleasa Pancreática/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Platelet and megakaryocyte endocytosis is important for loading certain granule cargo (ie, fibrinogen [Fg] and vascular endothelial growth factor); however, the mechanisms of platelet endocytosis and its functional acute effects are understudied. Adenosine 5'-diphosphate-ribosylation factor 6 (Arf6) is a small guanosine triphosphate-binding protein that regulates endocytic trafficking, especially of integrins. To study platelet endocytosis, we generated platelet-specific Arf6 knockout (KO) mice. Arf6 KO platelets had less associated Fg suggesting that Arf6 affects αIIbß3-mediated Fg uptake and/or storage. Other cargo was unaffected. To measure Fg uptake, mice were injected with biotinylated- or fluorescein isothiocyanate (FITC)-labeled Fg. Platelets from the injected Arf6 KO mice showed lower accumulation of tagged Fg, suggesting an uptake defect. Ex vivo, Arf6 KO platelets were also defective in FITC-Fg uptake and storage. Immunofluorescence analysis showed initial trafficking of FITC-Fg to a Rab4-positive compartment followed by colocalization with Rab11-positive structures, suggesting that platelets contain and use both early and recycling endosomes. Resting and activated αIIbß3 levels, as measured by flow cytometry, were unchanged; yet, Arf6 KO platelets exhibited enhanced spreading on Fg and faster clot retraction. This was not the result of alterations in αIIbß3 signaling, because myosin light-chain phosphorylation and Rac1/RhoA activation were unaffected. Consistent with the enhanced clot retraction and spreading, Arf6 KO mice showed no deficits in tail bleeding or FeCl3-induced carotid injury assays. Our studies present the first mouse model for defining the functions of platelet endocytosis and suggest that altered integrin trafficking may affect the efficacy of platelet function.
Asunto(s)
Factores de Ribosilacion-ADP/fisiología , Plaquetas/fisiología , Endocitosis/fisiología , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/metabolismo , Factor 6 de Ribosilación del ADP , Factores de Ribosilacion-ADP/deficiencia , Factores de Ribosilacion-ADP/genética , Animales , Biotinilación , Plaquetas/ultraestructura , Membrana Celular/metabolismo , Tamaño de la Célula , Retracción del Coagulo , Gránulos Citoplasmáticos , Fibrinógeno/metabolismo , Humanos , Ratones , Ratones Noqueados , Ratones Transgénicos , Transporte de Proteínas/fisiología , Transducción de Señal/fisiologíaRESUMEN
The GTPase-activating protein (GAP) specific to the small GTPase Arf6, ACAP3, is known to regulate morphogenesis of neurons in vitro. However, physiological significance of ACAP3 in the brain development in vivo remains unclear. Here, we show that ACAP3 is involved in neuronal migration in the developing cerebral cortex of mice. Knockdown of ACAP3 in the developing cortical neurons of mice in utero significantly abrogated neuronal migration in the cortical layer, which was restored by ectopic expression of wild type of ACAP3, but not by its GAP-inactive mutant. Furthermore, morphological changes of neurons during migration in the cortical layer were impeded in ACAP3-knocked-down cortical neurons. These results provide evidence that ACAP3 plays a crucial role in migration of cortical neurons by regulating their morphological change during development of cerebral cortex.
Asunto(s)
Factores de Ribosilacion-ADP/metabolismo , Movimiento Celular , Corteza Cerebral/embriología , Proteínas Activadoras de GTPasa/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Neuronas/citología , Factor 6 de Ribosilación del ADP , Animales , Células Cultivadas , Corteza Cerebral/citología , Corteza Cerebral/metabolismo , Femenino , Proteínas Activadoras de GTPasa/análisis , Proteínas Activadoras de GTPasa/genética , Técnicas de Silenciamiento del Gen , Proteínas de Transporte de Membrana/análisis , Proteínas de Transporte de Membrana/genética , Ratones Endogámicos C57BL , Ratones Endogámicos ICR , Mutación , Neuronas/metabolismoRESUMEN
ACAP3 (ArfGAP with coiled-coil, ankyrin repeat and pleckstrin homology domains 3) belongs to the ACAP family of GAPs (GTPase-activating proteins) for the small GTPase Arf (ADP-ribosylation factor). However, its specificity to Arf isoforms and physiological functions remain unclear. In the present study, we demonstrate that ACAP3 plays an important role in neurite outgrowth of mouse hippocampal neurons through its GAP activity specific to Arf6. In primary cultured mouse hippocampal neurons, knockdown of ACAP3 abrogated neurite outgrowth, which was rescued by ectopically expressed wild-type ACAP3, but not by its GAP activity-deficient mutant. Ectopically expressed ACAP3 in HEK (human embryonic kidney)-293T cells showed the GAP activity specific to Arf6. In support of this observation, the level of GTP-bound Arf6 was significantly increased by knockdown of ACAP3 in hippocampal neurons. In addition, knockdown and knockout of Arf6 in mouse hippocampal neurons suppressed neurite outgrowth. These results demonstrate that ACAP3 positively regulates neurite outgrowth through its GAP activity specific to Arf6. Furthermore, neurite outgrowth suppressed by ACAP3 knockdown was rescued by expression of a fast cycle mutant of Arf6 that spontaneously exchanges guanine nucleotides on Arf6, but not by that of wild-type, GTP- or GDP-locked mutant Arf6. Thus cycling between active and inactive forms of Arf6, which is precisely regulated by ACAP3 in concert with a guanine-nucleotide-exchange factor(s), seems to be required for neurite outgrowth of hippocampal neurons.
Asunto(s)
Factores de Ribosilacion-ADP/metabolismo , Proteínas Activadoras de GTPasa/fisiología , Hipocampo/metabolismo , Proteínas de Transporte de Membrana/fisiología , Neuritas , Neuronas/metabolismo , Factor 6 de Ribosilación del ADP , Animales , Hipocampo/citología , Ratones , Neuronas/citologíaRESUMEN
A small GTPase, Arf6, is involved in cytokinesis by localizing to the Flemming body (the midbody). However, it remains unknown how Arf6 contributes to cytokinesis. Here, we demonstrate that Arf6 directly interacts with mitotic kinesin-like protein 1 (MKLP1), a Flemming body-localizing protein essential for cytokinesis. The crystal structure of the Arf6-MKLP1 complex reveals that MKLP1 forms a homodimer flanked by two Arf6 molecules, forming a 2:2 heterotetramer containing an extended ß-sheet composed of 22 ß-strands that spans the entire heterotetramer, suitable for interaction with a concave membrane surface at the cleavage furrow. We show that, during cytokinesis, Arf6 is first accumulated around the cleavage furrow and, prior to abscission, recruited onto the Flemming body via interaction with MKLP1. We also show by structure-based mutagenesis and siRNA-mediated knockdowns that the complex formation is required for completion of cytokinesis. A model based on these results suggests that the Arf6-MKLP1 complex plays a crucial role in cytokinesis by connecting the microtubule bundle and membranes at the cleavage plane.
Asunto(s)
Factores de Ribosilacion-ADP/metabolismo , Citocinesis , Proteínas Asociadas a Microtúbulos/metabolismo , Factor 6 de Ribosilación del ADP , Factores de Ribosilacion-ADP/química , Factores de Ribosilacion-ADP/genética , Animales , Cristalografía por Rayos X , Células HeLa , Humanos , Ratones , Proteínas Asociadas a Microtúbulos/química , Proteínas Asociadas a Microtúbulos/genética , Mutación , Unión ProteicaRESUMEN
Plasma membrane proteins that enter cells by clathrin-independent endocytosis (CIE) are sorted either to lysosomes for degradation or recycled back to the plasma membrane. Expression of some MARCH E3 ubiquitin ligases promotes trafficking of CIE cargo proteins to lysosomes by ubiquitylating the proteins. Here, we show that co-expression of the ubiquitin-specific protease TRE17/USP6 counteracts the MARCH-dependent targeting of CIE cargo proteins, but not that of transferrin receptor, to lysosomes, leading to recovery of the stability and cell surface level of the proteins. The ubiquitylation of CIE cargo proteins by MARCH8 was reversed by TRE17, suggesting that TRE17 leads to deubiquitylation of CIE cargo proteins. The effects of TRE17 were dependent on its deubiquitylating activity and expression of TRE17 alone led to a stabilization of surface major histocompatibility complex class I (MHCI) molecules, a CIE cargo, suggesting that deubiquitylation of endogenous CIE cargo proteins promotes their stability. This study demonstrates that cycles of ubiquitylation and deubiquitylation can determine whether CIE cargo proteins are degraded or recycled.
Asunto(s)
Clatrina/metabolismo , Endocitosis/fisiología , Proteínas Proto-Oncogénicas/metabolismo , Ubiquitina Tiolesterasa/metabolismo , Clatrina/genética , Endocitosis/genética , Técnica del Anticuerpo Fluorescente , Células HeLa , Humanos , Transporte de Proteínas/genética , Transporte de Proteínas/fisiología , Proteínas Proto-Oncogénicas/genética , Ubiquitina Tiolesterasa/genética , UbiquitinaciónRESUMEN
The ability to perceive noxious stimuli is critical for an animal's survival in the face of environmental danger, and thus pain perception is likely to be under stringent evolutionary pressure. Using a neuronal-specific RNAi knock-down strategy in adult Drosophila, we recently completed a genome-wide functional annotation of heat nociception that allowed us to identify α2δ3 as a novel pain gene. Here we report construction of an evolutionary-conserved, system-level, global molecular pain network map. Our systems map is markedly enriched for multiple genes associated with human pain and predicts a plethora of novel candidate pain pathways. One central node of this pain network is phospholipid signaling, which has been implicated before in pain processing. To further investigate the role of phospholipid signaling in mammalian heat pain perception, we analysed the phenotype of PIP5Kα and PI3Kγ mutant mice. Intriguingly, both of these mice exhibit pronounced hypersensitivity to noxious heat and capsaicin-induced pain, which directly mapped through PI3Kγ kinase-dead knock-in mice to PI3Kγ lipid kinase activity. Using single primary sensory neuron recording, PI3Kγ function was mechanistically linked to a negative regulation of TRPV1 channel transduction. Our data provide a systems map for heat nociception and reinforces the extraordinary conservation of molecular mechanisms of nociception across different species.
Asunto(s)
Drosophila , Redes Reguladoras de Genes , Dolor Nociceptivo , Fosfolípidos , Transducción de Señal , Animales , Capsaicina/toxicidad , Fosfatidilinositol 3-Quinasa Clase Ib/genética , Fosfatidilinositol 3-Quinasa Clase Ib/fisiología , Drosophila/genética , Drosophila/fisiología , Calor , Humanos , Hipersensibilidad/genética , Ratones , Neuronas Aferentes/metabolismo , Dolor Nociceptivo/inducido químicamente , Dolor Nociceptivo/genética , Dolor Nociceptivo/fisiopatología , Fosfolípidos/genética , Fosfolípidos/metabolismo , Fosfolípidos/fisiología , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Fosfotransferasas (Aceptor de Grupo Alcohol)/fisiología , Canales Catiónicos TRPV/genética , Canales Catiónicos TRPV/metabolismo , Canales Catiónicos TRPV/fisiologíaRESUMEN
Activation of receptor tyrosine kinases leads to the formation of two different types of plasma membrane structures: peripheral ruffles and dorsal ruffles. Although the formation of both ruffle types requires activation of the small GTPase Rac, the difference in kinetics suggests that a distinct regulatory mechanism operates for their ruffle formation. DOCK1 and DOCK5 are atypical Rac activators and are both expressed in mouse embryonic fibroblasts (MEFs). We found that although PDGF-induced Rac activation and peripheral ruffle formation were coordinately regulated by DOCK1 and DOCK5 in MEFs, DOCK1 deficiency alone impaired dorsal ruffle formation in MEFs. Unlike DOCK5, DOCK1 bound to phosphatidic acid (PA) through the C-terminal polybasic amino acid cluster and was localized to dorsal ruffles. When this interaction was blocked, PDGF-induced dorsal ruffle formation was severely impaired. In addition, we show that phospholipase D, an enzyme that catalyzes PA synthesis, is required for PDGF-induced dorsal, but not peripheral, ruffle formation. These results indicate that the phospholipase D-PA axis selectively controls dorsal ruffle formation by regulating DOCK1 localization.
Asunto(s)
Estructuras de la Membrana Celular/metabolismo , Ácidos Fosfatidicos/fisiología , Proteínas de Unión al GTP rac/metabolismo , Secuencia de Aminoácidos , Animales , Células Cultivadas , Secuencia Conservada , Activación Enzimática , Factores de Intercambio de Guanina Nucleótido/metabolismo , Factores de Intercambio de Guanina Nucleótido/fisiología , Ratones , Ratones Transgénicos , Microscopía Fluorescente , Datos de Secuencia Molecular , Ácidos Fosfatidicos/metabolismo , Fosfolipasa D/metabolismo , Factor de Crecimiento Derivado de Plaquetas/fisiología , Estructura Terciaria de Proteína , Transporte de Proteínas , Proteínas Tirosina Quinasas/metabolismo , Transducción de Señal , Proteínas de Unión al GTP rac/genética , Proteínas de Unión al GTP rac/fisiologíaRESUMEN
Phosphatidylinositol (PI) 4,5-bisphosphate (PIP(2)), generated by PI 4-phosphate 5-kinase (PIP5K), regulates many critical cellular events. PIP(2) is also known to mediate plasma membrane localization of the Toll/IL-1 receptor domain-containing adaptor protein (TIRAP), required for the MyD88-dependent Toll-like receptor (TLR) 4 signaling pathway. Microglia are the primary immune competent cells in brain tissue, and TLR4 is important for microglial activation. However, a functional role for PIP5K and PIP(2) in TLR4-dependent microglial activation remains unclear. Here, we knocked down PIP5Kα, a PIP5K isoform, in a BV2 microglial cell line using stable expression of lentiviral shRNA constructs or siRNA transfection. PIP5Kα knockdown significantly suppressed induction of inflammatory mediators, including IL-6, IL-1ß, and nitric oxide, by lipopolysaccharide. PIP5Kα knockdown also attenuated signaling events downstream of TLR4 activation, including p38 MAPK and JNK phosphorylation, NF-κB p65 nuclear translocation, and IκB-α degradation. Complementation of the PIP5Kα knockdown cells with wild type but not kinase-dead PIP5Kα effectively restored the LPS-mediated inflammatory response. We found that PIP5Kα and TIRAP colocalized at the cell surface and interacted with each other, whereas kinase-dead PIP5Kα rendered TIRAP soluble. Furthermore, in LPS-stimulated control cells, plasma membrane PIP(2) increased and subsequently declined, and TIRAP underwent bi-directional translocation between the membrane and cytosol, which temporally correlated with the changes in PIP(2). In contrast, PIP5Kα knockdown that reduced PIP(2) levels disrupted TIRAP membrane targeting by LPS. Together, our results suggest that PIP5Kα promotes TLR4-associated microglial inflammation by mediating PIP(2)-dependent recruitment of TIRAP to the plasma membrane.
Asunto(s)
Fosfotransferasas (Aceptor de Grupo Alcohol)/fisiología , Receptor Toll-Like 4/metabolismo , Animales , Línea Celular , Membrana Celular/metabolismo , Ensayo de Inmunoadsorción Enzimática/métodos , Células HEK293 , Células HeLa , Humanos , Inflamación , Interleucina-1/metabolismo , Glicoproteínas de Membrana/metabolismo , Ratones , Microglía/metabolismo , Óxido Nítrico/química , Receptores de Interleucina-1/metabolismo , Transducción de Señal , Factores de TiempoRESUMEN
Proper neutrophil migration into inflammatory sites ensures host defense without tissue damage. Phosphoinositide 3-kinase (PI(3)K) and its lipid product phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P(3)) regulate cell migration, but the role of PtdIns(3,4,5)P(3)-degrading enzymes in this process is poorly understood. Here, we show that Src homology 2 (SH2) domain-containing inositol-5-phosphatase 1 (SHIP1), a PtdIns(3,4,5)P(3) phosphatase, is a key regulator of neutrophil migration. Genetic inactivation of SHIP1 led to severe defects in neutrophil polarization and motility. In contrast, loss of the PtdIns(3,4,5)P(3) phosphatase PTEN had no impact on neutrophil chemotaxis. To study PtdIns(3,4,5)P(3) metabolism in living primary cells, we generated a novel transgenic mouse (AktPH-GFP Tg) expressing a bioprobe for PtdIns(3,4,5)P(3.) Time-lapse footage showed rapid, localized binding of AktPH-GFP to the leading edge membrane of chemotaxing ship1(+/+)AktPH-GFP Tg neutrophils, but only diffuse localization in ship1(-/-)AktPH-GFP Tg neutrophils. By directing where PtdIns(3,4,5)P(3) accumulates, SHIP1 governs the formation of the leading edge and polarization required for chemotaxis.
Asunto(s)
Movimiento Celular , Polaridad Celular , Quimiotaxis , Fosfatos de Fosfatidilinositol/metabolismo , Monoéster Fosfórico Hidrolasas/fisiología , Animales , Células Cultivadas , Fosfatidilinositol 3-Quinasa Clase Ib , Humanos , Inositol Polifosfato 5-Fosfatasas , Isoenzimas/metabolismo , Isoenzimas/fisiología , Macrófagos/fisiología , Ratones , Ratones Transgénicos , Modelos Biológicos , Neutrófilos/fisiología , Proteína Oncogénica v-akt/genética , Proteína Oncogénica v-akt/metabolismo , Fosfohidrolasa PTEN/genética , Fosfohidrolasa PTEN/fisiología , Fosfatidilinositol 3-Quinasas/metabolismo , Fosfatidilinositol 3-Quinasas/fisiología , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatasas , Monoéster Fosfórico Hidrolasas/genética , Monoéster Fosfórico Hidrolasas/metabolismoRESUMEN
The lipid kinase phosphatidylinositol 4-phosphate 5-kinase (PIP5K) produces a versatile signaling phospholipid, phosphatidylinositol 4,5-bisphosphate. Three PIP5K isozymes, PIP5K1A, PIP5K1B, and PIP5K1C, have been identified in mammals so far. Although the functions of these three PIP5K isozymes have been extensively studied in vitro, the in vivo physiological roles of these PIP5K isozymes remain largely unknown. In this study, we examined the functions of PIP5K1A and PIP5K1B in spermatogenesis, using Pip5k1a-knockout (KO), Pip5k1b-KO, and Pip5k1a/Pip5k1b double (D)-KO mice. Pip5k1a-KO and D-KO males were subfertile and completely sterile, respectively. F-actin in the seminiferous epithelium was disorganized in the D-KO mice, although F-actin bundles at the apical ectoplasmic specialization was not affected. D-KO seminiferous tubules contained a greatly decreased number of elongated spermatids. Flagella of sperm from Pip5k1a-KO and D-KO mice remarkably underwent morphological change, whereas Pip5k1b-KO sperm were morphologically normal. Notably, the flagellar shape of D-KO sperm was more severely impaired than that of Pip5k1a-KO sperm. These results suggest that PIP5K1A and PIP5K1B may coordinately and/or redundantly function in the maintenance of sperm number and morphology during spermatogenesis.
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Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Espermatogénesis/fisiología , Actinas/fisiología , Animales , Isoenzimas/genética , Isoenzimas/metabolismo , Masculino , Ratones , Ratones Noqueados , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Epitelio Seminífero/metabolismo , Túbulos Seminíferos/metabolismo , Cola del Espermatozoide/metabolismo , Espermátides/metabolismoRESUMEN
Developmental timing in the nematode Caenorhabditis elegans is controlled by heterochronic genes, mutations in which cause changes in the relative timing of developmental events. One of the heterochronic genes, let-7, encodes a microRNA that is highly evolutionarily conserved, suggesting that similar genetic pathways control developmental timing across phyla. Here we report that the nuclear receptor nhr-25, which belongs to the evolutionarily conserved fushi tarazu-factor 1/nuclear receptor NR5A subfamily, interacts with heterochronic genes that regulate the larva-to-adult transition in C. elegans. We identified nhr-25 as a regulator of apl-1, a homolog of the Alzheimer's amyloid precursor protein-like gene that is downstream of let-7 family microRNAs. NHR-25 controls not only apl-1 expression but also regulates developmental progression in the larva-to-adult transition. NHR-25 negatively regulates the expression of the adult-specific collagen gene col-19 in lateral epidermal seam cells. In contrast, NHR-25 positively regulates the larva-to-adult transition for other timed events in seam cells, such as cell fusion, cell division and alae formation. The genetic relationships between nhr-25 and other heterochronic genes are strikingly varied among several adult developmental events. We propose that nhr-25 has multiple roles in both promoting and inhibiting the C. elegans heterochronic gene pathway controlling adult differentiation programs.
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Caenorhabditis elegans/genética , Caenorhabditis elegans/fisiología , Receptores Citoplasmáticos y Nucleares/metabolismo , Animales , Caenorhabditis/genética , Caenorhabditis/metabolismo , Caenorhabditis elegans/metabolismo , Diferenciación Celular/genética , División Celular , Redes Reguladoras de Genes , Larva/genética , Larva/metabolismo , MicroARNs/genética , MicroARNs/metabolismo , Mutación , Receptores Citoplasmáticos y Nucleares/genéticaRESUMEN
The predominant pathway for phosphatidylinositol (4,5)-bisphosphate (PI(4,5)P(2)) synthesis is thought to be phosphorylation of phosphatidylinositol 4-phosphate at the 5 position of the inositol ring by type I phosphatidylinositol phosphate kinases (PIPK): PIPKIalpha, PIPKIbeta, and PIPKIgamma. PIPKIgamma has been shown to play a role in PI(4,5)P(2) synthesis in brain, and the absence of PIPKIgamma is incompatible with postnatal life. Conversely, mice lacking PIPKIalpha or PIPKIbeta (isoforms are referred to according to the nomenclature of human PIPKIs) live to adulthood, although functional effects in specific cell types are observed. To determine the contribution of PIPKIalpha and PIPKIbeta to PI(4,5)P(2) synthesis in brain, we investigated the impact of disrupting multiple PIPKI genes. Our results show that a single allele of PIPKIgamma, in the absence of both PIPKIalpha and PIPKIbeta, can support life to adulthood. In addition, PIPKIalpha alone, but not PIPKIbeta alone, can support prenatal development, indicating an essential and partially overlapping function of PIPKIalpha and PIPKIgamma during embryogenesis. This is consistent with early embryonic expression of PIPKIalpha and PIPKIgamma but not of PIPKIbeta. PIPKIbeta expression in brain correlates with neuronal differentiation. The absence of PIPKIbeta does not impact embryonic development in the PIPKIgamma knock-out (KO) background but worsens the early postnatal phenotype of the PIPKIgamma KO (death occurs within minutes rather than hours). Analysis of PIP(2) in brain reveals that only the absence of PIPKIgamma significantly impacts its levels. Collectively, our results provide new evidence for the dominant importance of PIPKIgamma in mammals and imply that PIPKIalpha and PIPKIbeta function in the generation of specific PI(4,5)P(2) pools that, at least in brain, do not have a major impact on overall PI(4,5)P(2) levels.
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Encéfalo/enzimología , Diferenciación Celular , Embrión de Mamíferos/enzimología , Neuronas/enzimología , Fosfatidilinositol 3-Quinasas/metabolismo , Fosfatidilinositol 4,5-Difosfato/biosíntesis , Animales , Encéfalo/embriología , Química Encefálica/genética , Embrión de Mamíferos/embriología , Desarrollo Embrionario/genética , Humanos , Ratones , Ratones Noqueados , Neuronas/metabolismo , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 4,5-Difosfato/genéticaRESUMEN
Pertussis toxin (PTX) is recognized as a specific tool that uncouples receptors from G(i) and G(o) through ADP-ribosylation. During the study analyzing the effects of PTX on Ang II type 1 receptor (AT1R) function in cardiac fibroblasts, we found that PTX increases the number of AT1Rs and enhances AT1R-mediated response. Microarray analysis revealed that PTX increases the induction of interleukin (IL)-1beta among cytokines. Inhibition of IL-1beta suppressed the enhancement of AT1R-mediated response by PTX. PTX increased the expression of IL-1beta and AT1R through NF-kappaB, and a small GTP-binding protein, Rac, mediated PTX-induced NF-kappaB activation through NADPH oxidase-dependent production of reactive oxygen species. PTX induced biphasic increases in Rac activity, and the Rac activation in a late but not an early phase was suppressed by IL-1beta siRNA, suggesting that IL-1beta-induced Rac activation contributes to the amplification of Rac-dependent signaling induced by PTX. Furthermore, inhibition of TLR4 (Toll-like receptor 4) abolished PTX-induced Rac activation and enhancement of AT1R function. However, ADP-ribosylation of G(i)/G(o) by PTX was not affected by inhibition of TLR4. Thus, PTX binds to two receptors; one is TLR4, which activates Rac, and another is the binding site that is required for ADP-ribosylation of G(i)/G(o).
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Angiotensina I/metabolismo , Toxina del Pertussis/farmacología , Receptores de Angiotensina/efectos de los fármacos , Receptores Toll-Like/fisiología , Regulación hacia Arriba/efectos de los fármacos , Proteínas de Unión al GTP rac/metabolismo , Animales , Células Cultivadas , Interleucina-1/biosíntesis , Miocardio/citología , Miocardio/metabolismo , Ratas , Ratas Sprague-Dawley , Especies Reactivas de Oxígeno/metabolismo , Receptores de Angiotensina/metabolismoRESUMEN
The phospholipid kinase phosphatidylinositol 4-phosphate 5-kinase (PIP5K) catalyzes the phosphorylation of the membrane phospholipid phosphatidylinositol 4-phosphate to generate the pleiotropic phospholipid phosphatidylinositol 4,5-bisphosphate [PI(4,5)P(2) ]. To date, three mammalian PIP5K isozymes, α, ß, and γ, and several splicing variants of the γ isozyme have been identified. These PIP5K isozymes and PIP5Kγ variants play critical roles in various cellular functions through their product PI(4,5)P(2) . The small GTPase Arf6 is one of the key activators of PIP5K. Increasing evidence suggests that PIP5K functions as a downstream effector of Arf6 to regulate a wide variety of cellular functions, such as exocytosis, endocytosis, endosomal recycling, membrane ruffle formation, immune response, and bacterial invasion. In this review, we place our focus on the recent advances in Arf6/PIP5K signaling and its linkage to cellular functions.